bluuebunny/crossref_metadata_2025 huggingface dataset:
Vidicon Spectrometer Applied to Simultaneous Enzyme Determinations
Authors: Milano Michael J , Pardue Harry L
Published: January 2020

Lactate dehydrogenase and alkaline phosphatase activities in the same medium can be determined simultaneously, at 350 and 550 nm, with a vidicon spectrometer. Substrate concentrations and pH have been made optimum for the combined analysis. These conditions result in activities for lactate dehydrogenase that are equivalent to those found by methods in common use, and activities for alkaline phosphatase that are about 31% below the maximum values that could be obtained with its substrate used at the same pH and temperature in the absence of NAD+ and lactate. However, activities measured by the simultaneous analysis were proportional to those obtained by other methods used in clinical laboratories, and the coefficients of variation were 2.3% for lactate dehydrogenase and 3% for alkaline phosphatase.
Effects on Radioimmunoassay of Digoxin of Varying Incubation Periods for Antigen—Antibody Reaction and Varying Periods of Adsorption by Dextran-Coated Charcoal
Authors: Kuno-Sakai Harumi , Sakai Hideto
Published: January 2020

In radioimmunoassay of digoxin by the dextran-coated charcoal method, duration of the incubation period for the antigen-antibody reaction and time allowed for dextran-coated charcoal to adsorb the free antigen are critical factors. We compared effects of prolonged periods of adsorption on results obtained with five commercial kits. In all cases, the digoxin concentrations increased linearly with adsorption time, but the increase varied among kits. Kallestad and New England Nuclear 3H-kits and the Squibb 125I-kit showed less increase (i.e., gave relatively consistent results after various periods of adsorption); the Schwarz/Mann 125I-kit and Clinical Assays 3H-kit showed greater increases. Precautionary steps are outlined for radioimmunoassays by this technique.
Statistical Evaluation of Method-Comparison Data
Authors: Wu Gaw Tzu , Twomey Stanley L , Thiers Ralph E
Published: January 2020

Comparison of two methods for determining a substance is a common procedure in clinical chemistry. Almost without exception, normal theory statistics are used with the tacit assumption that the data under test show gaussian distributions. Experience has led us to question this assumption for populations of differences between values obtained by two methods applied to common samples and for populations of paired values for each method as used in correlation analysis. Data are presented from our laboratory to illustrate these points, as well as an examination of data from relevant papers that appeared in this journal from 1968 through 1973. These examples indicate that incorrect interpretation of results because of insufficient attention to nongaussian distribution is a real and present danger. We present a systematic approach to the statistical aspect of comparing methods that enables one to avoid this problem.
Automated Differentiation and Measurement of Hexosaminidase Isoenzymes in Biological Fluids and Its Application to Preand Postnatal Detection of Tay—Sachs Disease
Authors: Salfer Abraham , Parkhurst George W , Amoroso June
Published: January 2020

Three hexosaminidase (EC 3.2.1.52) isoenzymes other than isoenzymes A and B in body fluids have been separated by chromatography on diethylaminoethyl cellulose. By inserting a microcolumn into a continuous-flow system for automated, fluorometric hexosaminidase analysis [Clin. Chem. 20, 538 (1974)], samples eluted with buffered-NaCl gradients can be continuously monitored. Isoenzyme patterns were obtained for fluids from normal individuals, pregnant women, Tay—Sachs disease carriers, pregnant carriers, and patients with the disease. These chromatograms revealed a hitherto undetected isoenzyme (I3) in serum. An increase in serum hexosaminidase isoenzyme I2(or P) during pregnancy is characteristic of a carrier pattern. Our data show that serum and urinary hexosaminidase isoenzyme patterns may be used in addition to leukocyte analysis, to distinguish a pregnant carrier from a normal pregnant woman. All fluids tested demonstrated no isoenzyme A activity and above-normal activity of isoenzymes B and (or) I2 in homozygotes. Urine is preferred fluid for postnatal and amniotic fluid for the prenatal diagnosis of the disease. Quantitative data on isoenzyme A obtained with the procedure described here agree well with those obtained by heatand pH-inactivation methods.
Determination of Orotic and Dihydroorotic Acids in Biological Fluids and Tissues
Authors: Kesner Leo , Aronson Frank L , Silverman Morris , Chan Phillip C
Published: January 2020

In commonly used procedures for colorimetric assay of orotic acid in biological materials, certain substances interfere, positively or negatively. Some that interfere negatively are ornithine, cysteine, citrulline, tyrosine, proline, hydroxyproline, and polyamines. Protein and interfering agents are easily removed by first isolating the total organic acid fraction. The orotic acid in this fraction can then be determined by an established colorimetric procedure. A rapid liquid—liquid column-chromatographic procedure is described, if analysis of both dihydroorotic and orotic acid is required.
Comparison of Clinical Assays for Serum Corticosteroids
Authors: Jiang Nai-Siang , Machacek Dwaine , Wadel Orlando P
Published: January 2020

We compared results obtained by (a) chemical (spectrophotometric) and competitive protein-binding assays for serum corticosteroids, and (b) competitive proteinbinding and radioimmunoassay, with and without CCl4 extraction, for serum cortisol. The two corticosteroid methods gave identical results. Radioimmunoassay with and without a CCl4 extraction step gave identical results for cortisol. Competitive protein-binding assays for cortisol (with and without CCl4 extraction) and radioimmunoassay for cortisol gave similar results. Specimens with above-normal corticosterone or 11-deoxycortisol concentrations are an exception: only methods that included CCl4 extraction gave correct values for cortisol; methods without such an extraction step overestimated cortisol. The normal serum corticosteroid concentration (mean ± SD) was 175 ± 53 µg/liter for morning specimens, and 103 ± 40 µg/liter for specimens collected in the afternoon. For cortisol, the corresponding values were 162 ± 44 and 82 ± 29 µg/liter.
Effect of Albumin on Serum Digoxin Radioimmunoassays
Authors: Voshall David L , Hunter Lynda , Grady Harold J
Published: January 2020

The use of certain commercially available radioimmunoassay kits for measurement of digoxin results in errors in the determined digoxin concentration of patients when these kits are used according to manufacturers' directions. One factor that is, in part, responsible for these errors is a difference between standards and samples with respect to albumin concentration. Three of the four kits investigated showed a significant inverse relationship between the albumin concentration in the sample and the binding of radiolabeled digoxin by its antibody when the albumin concentration was varied over an extended range. It is apparent, however, that differences in albumin concentration do not completely explain the observed variations in the assay values.
Automated Method for Determination of Bound N-Acetylneuraminic Acid in Serum
Authors: Rey Elisabeth , Gerbaut Lucile , Lombart Christian
Published: January 2020

A modified resorcinol method for sialic acid determination has been successfully adapted to the Technicon AutoAnalyzer. The present method requires only 25 µl of serum, and 30 samples can be analyzed per hour. It may be used to measure sialic acid in concentrations ranging from 10 to 100 mg/liter.
Mechanized Enzymatic Determination of Triglycerides in Serum
Authors: Bucolo Giovanni , Yabut Juanito , Chang Ting Yen
Published: January 2020

A procedure for enzymatic determination of serum triglycerides [Clin. Chem. 19, 476 (1973)] has been adapted for use in continuous-flow analysis (Technicon AutoAnalyzer). A very simple manifold is used; serum is incubated at 37 °C with the lipase and α-chymotrypsin in potassium phosphate buffer (0.1 mol/liter, pH 7, containing 1.50 g of bovine serum albumin per liter). The liberated glycerol is dialyzed against the complete glycerol reagent. The change in absorbance at 340 nm resulting from oxidation of NADH is proportional to the dialyzed glycerol. The same manifold can be used to determine preformed glycerol if the hydrolyzing enzymes are omitted. The lipase used need not be extensively purified because the dialyzer removes coloring material and contaminating enzymes that would interfere with the components of the glycerol reagent in the manual procedure. The hydrolysis is complete, as shown by the use of equivalent glycerol standards. No prior treatment of the samples is necessary. Assays are run at 60 per hour in the AutoAnalyzer I, 80 per hour in the AutoAnalyzer II. Results with both instruments for 150 samples correlated well with those obtained by the same enzymatic manual method and by the AutoAnalyzer fluorometric procedure.
Binding of 63Ni(II) to Ultrafiltrable Constituents of Rabbit Serum In Vivo and In Vitro
Authors: Asato Noritake , van Soestbergen Maria , Sunderman F William
Published: January 2020

Binding of 63Ni(Il) to ultrafiltrable constituents of rabbit serum was studied (a) after in vitro incubation (2 h, 37 °C) of rabbit serum with 63NiCl2 (10-100 µmol/liter), and (b) at intervals (0.25-2 h) after in vivo administration of 63NiCl2 (40-160 µmol/kg body wt, i.v.). Serum ultrafiltrates were fractionated by thin-layer chromatography, and the separated compounds made visible by autoradiography and by ninhydrin staining. Several (≃5) ultrafiltrable 63Ni-complexes were demonstrable as distinct radiodense 63Ni-bands with chromatographic mobilities corresponding to those of ninhydrin-positive bands. Unbound 63Ni(II) was not detected in serum ultrafiltrates in either the in vitro or in vivo experiments. In sera (n = 10) incubated in vitro with 63Ni(II) (10 µmol/ liter), the mean percentage of ultrafiltrable 63Ni was 36% (range = 33-38) of total serum 63Ni. In contrast, in sera (n = 10) obtained 2 h after i.v. injection of 63Ni(II) (40 µmol/kg), the mean concentration of total serum 63Ni was 10.8 µmol/liter (range = 6-14), and the mean percentage of ultrafiltrable 63Ni was 15% (range = 9-21) of total serum 63Ni. The disparity between the percentages of ultrafiltrable 63Ni obtained in vitro and in vivo was obviated when the in vivo experiments were performed in rabbits bilaterally nephrectomized, with ligated common bile ducts. This investigation confirms the existence of several nickel receptors in serum ultrafiltrates and substantiates the role of ultrafiltrable complexes in the excretion of nickel.
Intercomparison Program of Lead, Mercury, and Cadmium Analysis in Blood, Urine, and Aqueous Solutions
Authors: Lauwerys Robert , Buchet Jean-Pierre , Roels Harry , Berlin Alexander , Smeets Jan
Published: January 2020

Sixty-six European laboratories participated in an intercomparison program of lead, mercury, and cadmium analysis in blood, urine, and aqueous solutions. The experimental protocol was designed in such a way that the effect of precision, experience, and analytical method could be evaluated. For all the analyses, the scatter of the reported results is important. The major factor influencing the variability of the results is the intralaboratory variation. Analytical methods and degree of experience do not seem to have a significant influence. However, with the exception of mercury determination in urine, a satisfactory intralaboratory precision is not sufficient to make the interlaboratory variation acceptable. It appears that systematic errors are responsible for the high interlaboratory variation observed between "precise" laboratories.
Micromethod for Lead Determination in Whole Blood by Atomic Absorption, with Use of the Graphite Furnace
Authors: Fernandez Frank J
Published: January 2020

I describe a micro-scale method for determining lead in whole blood by utilizing a graphite furnace. Sample pretreatment consists of fivefold dilution with a dilute surfactant. The method is directly calibrated with lead standards prepared in dilute HNO3. To eliminate a small, nonspecific absorption signal from the blood matrix, simultaneous background correction is used. Interlaboratory comparison with a flame atomic absorption technique that requires extraction yielded high correlation (r = 0.98). Within-run precision (coefficient of variation) ranged from 2 to 4%. Lead in blood can be accurately measured in as little as 20 µl of blood, hence the method is suitable for routine laboratory use and for pediatric screening.
Microanalytical System for Determination of Picogram Quantities of Metals in Metalloenzymes, as Illustrated with Zinc-containing Enzymes
Authors: Kawaguchi Hiroshi , Auld David S
Published: January 2020

Microwave-induced emission spectrometry combined with gel-exclusion chromatography provides a microanalytical system capable of precisely measuring 10-10 to 10-13 g of metal in microgram amounts of enzyme. Such sensitivity greatly exceeds that of other, more conventional methods. Metal quenching agents and low-molecular-weight protein contaminants were removed from the enzyme by Sephadex G-100 chromatography in microbore columns (0.3 x 25 cm). Droplet fractions were analyzed for zinc by the present method, for enzyme activity, and for protein content. With this analytical system we could demonstrate that stoichiometric amounts of zinc are present in the RNA-dependent DNA polymerase, the reverse transcriptase, from woolly monkey type C RNA tumor virus. The precision of the method for zinc was demonstrated by the coefficient of variation of 4.4% for 10 µg of zinc per liter. Validity and accuracy of the method were established by determining zinc in a series of zinc metalloenzymes of known metal content and stoichiometry.
Effect of Carbon Cup Aging on Plasma Zinc Determination by Flameless Atomic Absorption Spectrometry
Authors: Chool Michael K , Todd John K , Boyd Nigel D
Published: January 2020

Determination of zinc in blood plasma by flameless atomic absorption spectrometry is discussed, with particular reference to the protocol required for the successful use of the Varian-Techtron Carbon Rod Atomizer. Cup aging is shown to be an important factor in limiting the precision of this analytical technique and ways of minimizing the problem are described. Matrix problems have also been encountered, which precluded the use of aqueous standard curves and the method of standard additions. We propose the use of plasma in preparing standard curves, the values for which are corrected for inherent plasma zinc, as a possible solution to the problem.
New, Simple Maltogenic Assay for Mechanized Determination of Alpha-Amylase Activity in Serum and Urine
Authors: Proelss Henning F , Wright Billy W
Published: January 2020

We report a new method for the mechanized determination of serum and urinary alpha-amylase by use of a continuous-flow system, based on the measurement of maltose formed by incubating the sample with amylodextrin at pH 7 and 40 °C. After dialysis, maltose is converted enzymatically to glucose, which is measured by Trinder's glucose oxidase—peroxidase method [J. Clin. Pathol. 22, 246 (1969)]. The reaction is linear for amylase activities up to 1400 Somogyi units/dl (2560 U/liter) and for maltose concentrations through 1500 mg/dl. No blank assay is required; consequently precision is improved and the automated system is simplified. Calibration with primary maltose standards increases accuracy and reliability. Common reducing substances in serum and urine do not interfere at their normal concentrations. There is a linear correlation between the results of this method and those of chromogenic and iodometric methods for normal and pathologic sera and urines. The chromogenic method yields significantly higher results and the iodometric method significantly lower results than this maltogenic method for elevated amylase activities. The normal range is 40-140 Somogyi units/dl (73-256 U/liter).
Fluorometric Assay of Serum γ-glutamyltransferase in Solution and on a Semi-Solid Surface
Authors: Rietz Bernd , Guilbault George G
Published: January 2020

We describe an enzymatic, fluorometric method for measuring the activity of serum γ-gIutamyItransferase (EC 2.3.2.2) in solution and on silicone rubber pads. N-γ-L-Glutamyl-α-naphthylamide in tris(hydroxymethyl)aminomethane buffer is used as substrate. In solution, measurements are performed at 37 °C with use of a reaction volume of 3.0 ml, in Pyrex cuvets. Measurements on silicone rubber pads are also made at 37 °C, after establishing a stable substrate film by lyophilizing the reagent for the γ-glutamyltransferase assay on the surface of the pads. Only 10-50 µl of buffered substrate and serum containing the enzyme to make a total volume of 60 µl are necessary for each assay. The rate of appearance of α-naphthylamine fluorescence (λex = 344 nm; λem = 445 nm), liberated from N-γ-L-glutamyl-α-naphthylamide by the enzymatic action of γ-glutamyltransferase, is measured and equated to its activity in serum. Calibration plots of the change in fluorescence per min vs. enzyme concentration for measurements in solution and on pads show a good proportionality in the range of 26.5-265 U/liter, and indicate the usefulness of these methods.
Nonchromatographic Radioimmunoassay of Plasma 11-Deoxycortisol, for Use in the Metyrapone Test, with Polyethylene Glycol as the Precipitant
Authors: Lee Lilian M Y , Schiller Harvey S
Published: January 2020

We have developed a simple, reliable radioimmunoassay for plasma 11-deoxycortisol. The method does not require chromatography but instead makes use of a simple extraction procedure which, in combination with the antibody characteristics, is highly specific for the metyrapone test. Polyethylene glycol was used to separate free and antibody-bound steroid. The smallest amount measurable is 15 pg (2.0 µg/ liter of plasma). The method is shown to be precise and accurate. Intra-assay precision of the method for two plasma pools was 26.7 ± 2.5 µg/ liter (CV = 9.4%) and 61.2 ± 3.7 µg/ liter (CV = 6.0%). The respective inter-assay precision was 27.0 ± 1.7 µg/ liter (CV = 6.3%) and 59.9 ± 2.3 µg/ liter (CV = 3.8%). The validity of the assay was further verified by evaluating the plasma 11-deoxycortisol response to metyrapone administration. The relative simplicity of the method and the commercial availability of all reagents, including antisera, makes this radioimmunoassay procedure practical for use in clinical laboratories.
Above-Normal Urinary Excretion of Urinary Ceramides in Farber's Disease, and Characterization of Their Components by High-Performance Liquid Chromatography
Authors: Iwamori Masao , Moser Hugo W
Published: January 2020

We compared the sphingolipid content of urine from a patient with Farber's disease with that of control urine. The ceramides were measured by high-performance liquid chromatography. The patient's urine contained 1.2 µg of ceramides per milligram of creatinine, more than 200-fold the normal amount. The urinary ceramides were isolated by high-performance liquid chromatography for further identification. They contained mainly nonhydroxy fatty acids and only a small quantity of those with 2-hydroxy fatty acids. This contrasts with the previously described composition of the patient's renal and cerebellar tissue. The fatty acid and long-chain base compositions of the urinary ceramides containing nonhydroxy fatty acids were nearly identical to those of the patient's kidney.
Fluorometry of Citrate in Serum, with Use of Citrate (pro-3S)-Lyase
Authors: Tomisek Arthur J , Winkler Edward M , Natelson Samuel
Published: January 2020

We describe a procedure for enzymatic assay of citrate in human serum. The citrate is degraded to acetate and oxaloacetate with citrate oxaloacetate-lyase (pro-3S-CH2 · COO- → acetate) (EC 4.1.3.6). Some oxaloacetate loses CO2 to form pyruvate. Addition of malate and lactate dehydrogenases (EC 1.1.1.37 and 1.1.1.27) permits determination of the oxaloacetate and pyruvate generated, and thus of the citrate concentration. The decrease in NADH concentration is measured fluorometrically. Results obtained for 30 consecutive human sera by this procedure were compared to the procedure in which the citrate is converted to pentabromoacetone. There was no statistically significant difference in values obtained by the two procedures. The range of values (mean ± 2 SD) found for sera from 25 blood donors by this procedure was 12.8-27.2 mg/liter (mean, 19.0 mg/ liter). Serum citrate as measured by both procedures during a glucose tolerance test was decreased from initial values under the influence of administered glucose (and endogenous insulin). Insulin concentrations were also measured during these glucose-tolerance tests. Citrate concentrations remain subnormal after the glucose and insulin concentrations return to their initial values. This accords with published reports.
Ultramicroscale Determination of Clinical Chemical Values for Blood during the First Four Days of Postnatal Life
Authors: deBaare Larissa , Lewis Jean , Sing Helen
Published: January 2020

Ultramicro procedures requiring 5-10 µl of serum or blood per analysis were used in determining blood constituents of healthy full-term newborns during the first four days of life. The resulting values appeared to be influenced by age, sex, and race. Values for total protein, albumin, urea nitrogen, and uric acid in serum decreased with time; serum inorganic phosphorus and whole-blood aldosaccharoses increased. Serum from females had higher values than that from males for total proteins, albumin, and inorganic phosphorus. The values for serum calcium and alkaline phosphatase were consistently higher in Negro than in white infants; values for uric acid were higher in the latter.
A More Sensitive Automated Method for Determination of Ornithine Carbamoyltransferase Activity in Human Serum
Authors: Clayson Kathleen J , Fine James S , Strandjord Paul E
Published: January 2020

Ornithine carbamoyltransferase (EC 2.1.3.3) activity is a sensitive, specific indicator of hepatocellular injury. This paper describes development of an improved automated procedure for measurement of this activity. Triethanolamine—ethylenedlaminetetraacetate is used as a buffer, and activity is determined by measuring the concentration of the product, citrulline. Kinetic studies have been performed to determine optimal pH and L-ornithine and carbamoyl phosphate concentrations. Recovery of citrulline was studied. The upper limit of normal obtained in a study of 106 blood-bank donors was 6 U/liter. The automated procedure developed as a result of these studies, in which optimal assay conditions are used, produces a threefold increase in sensitivity and permits use of a sample volume of 1 ml.
Electronic Timing of a High-Speed Immunonephelometric Continuous-Flow System
Authors: Lizana Jorge , Jansson Lennart , Hellsing Kristoffer
Published: January 2020

We describe a high-speed immunonephelometric continuous-flow system. The mechanical timing assembly and cams of the sampler were replaced by an external electronic control unit. Improved sampling precision, speeds of 2 to 900 samples per hour, and a wide variety of sample-to-wash ratios are the most important advantages of the unit. We analyzed albumin and fibrinogen at speeds of 150, 180, and 210 samples per hour. Carry-over studies showed that, in clinical practice, it should be possible to run the system at 150 samples per hour with an interaction of less than 5% and a precision (CV) of around 4%. Comparative studies with a previously described method, run at 60 samples per hour, showed correlation coefficients of 0.968 and 0.935 for albumin and fibrinogen, respectively. The electronic control unit can be used to increase speed and improve sampling precision of any continuous-flow system. An immunonephelometric system equipped with this device provided fast and reliable results, and required less antiserum than other systems described.
Improved Estimation of Urinary Myoglobulin by Counterimmunoelectrophoresis, as Compared with the Double Immunodiffusion Technique
Authors: Hibrawi Hazem , Blaker Robert G
Published: January 2020

We describe a simple, sensitive, and relatively inexpensive counterimmunoelectrophoretic technique for detection and estimation of myoglobin. The test can be completed within 90 min, compared with 12-48 h required by double immunodiffusion. With the counterimmunoelectrophoretic technique we could detect myoglobin in concentrations of 2-3 mg/liter, while the detection limit by double immunodiffusion was about 9 mg/liter.
Some Sources of Errors and Artifacts in Spectrophotometric Measurements
Authors: Ellis Keith J , Morrison John F
Published: January 2020

Some of the more important functions and limitations of spectrophotometers in relation to biochemical analyses, are described in nonmathematical terms. Possible sources of errors and artifacts are discussed, together with suggestions for avoiding or minimizing such effects.
Rapid Analysis for Theophylline in Serum by Use of High-Pressure Cation-Exchange Chromatography
Authors: Weinberger Miles , Chidsey Charles
Published: January 2020

We have developed a quantitative assay for serum theophylline by high-pressure cation-exchange chromatography. As little as 0.1-ml aliquots of serum were prepared for analysis simply by diluting them with a solution of the internal standard (8-chlorotheophylline). Theophylline and the internal standard were eluted in 17 and 27.5 min, respectively, and the peaks for them were distinct from those of other xanthines and uric acids. We encountered no interference when we used: (a) plasma obtained from blood anticoagulated with citrate; (b) hemolyzed, lipemic, or icteric serum; or (c) 52 samples from patients who were receiving various other medications. An analysis of calibration suggested that errors greater than ±2.6 mg/liter, a clinically acceptable range, were highly unlikely. Sensitivity was sufficient to identify less than 2.5 mg/liter. This assay was compared with the usual method involving solvent extraction and absorbance measurement in the ultraviolet and found to have similar accuracy, although it is easier, faster, requires less sample, and potentially is more specific.
Venous Serum, Capillary Serum, and Capillary Plasma Compared for Use in Determination of Lactate Dehydrogenase and Aspartate Aminotransferase Activities
Authors: Haymond Russell E , Knight Joseph A
Published: January 2020

Lactate dehydrogenase and aspartate aminotransferase activities in capillary serum or plasma were significantly greater than in simultaneously assayed venous serum, the greatest differences being between capillary and venous serum. Although some difference is attributable to tissue fluid contributions, platelets seem to account for most of it, with possible small contributions from leukocytes. Capillary tube size and type appear to be important factors. We recommend that when capillary blood is to be used for enzyme assay, it should be processed as plasma.
Electrophoretic Patterns of Alkaline Phosphatase Isoenzymes in Human Sera with Abnormally High Activity, and an Unusual Band Observed in Sera of Patients with Pancreatic Cancer
Authors: Cha Chung-Ja Mo , Mastrofrancesco Balduino , Cha Sungman , Randall Henry T
Published: January 2020

Alkaline phosphatase isoenzymes in sera were resolved by electrophoresis on cellulose acetate membranes into seven different bands (L1, B, Pl, L2, I1, I2, and Pa, in decreasing order of electrophoretic mobility). The slowest-moving band (Pa) was observed in the sera of 16 patients—15 with cancer of the pancreas and one with hemochromatosis. Sera of 50 other patients with malignant or benign diseases did not show the Pa band. The Pa band is more heat labile than is the liver isoenzyme (L1). Its behavior toward inhibitors (L-phenylalanine and L-homoarginine) is similar to that of L1. Sera containing the Pa band exhibit a diffuse band in the region where isoenzymes of intestinal origin migrate; however, its heat stability and stereospecific inhibition are different from those of intestinal isoenzymes in sera that show no Pa band.
Evaluation of the Beckman "System TR Enzyme Analyzer"
Authors: Passey Richard , Gillum Ronald L , Giles Mary Louise , Fuller John B
Published: January 2020

We evaluated 16 claims made by Beckman Instruments, Inc. for its Enzyme Analyzer (System TR), under a rigid written protocol for the Product Evaluation Subcommittee of the Standards Committee of the College of American Pathologists. We found the following to be within the company's specifications: (a) accuracy and precision of the temperature control; (b) accuracy and precision of the sample and reagent pipets; (c) instrument precision, both within-run and between-day; (d) carry-over from a sample with activity > 1000 U/liter; (e) instrument-to-instrument variation; (f) analytical linearity; (g) analysis time; (h) correlation of the instrumentprinted answer with the activity calculated manually from a strip-chart recorder; (i) precision of the instrument's built-in electronic "standard"; (j) effectiveness of the over-range indicators; and (k) correlation between results of these enzyme assay methods and those for kinetic methods used in our laboratory. The instrument performed well.
New Colorimetric Reaction for End-Point, Continuous-Flow, and Kinetic Measurement of Urea
Authors: Jung David , Biggs Homer , Erikson John , Ledyard Peggy U
Published: January 2020

A reaction of urea, o-phthalaldehyde and N-(1-naphthyl)ethylenediamine is described for measurement of urea by manual, continuous-flow, and kinetic methods. The continuous-flow system requires 25 µl of sample; 40 samples can be analyzed per hour. The kinetic method requires no enzymes, has no lag phase, and has good sensitivity. A major advantage of the reaction is that it occurs at a temperature of 37 °C or lower. The results obtained by all three methods agree well with those for a continuous-flow procedure in which diacetyl is a reagent.
Improved Colorimetric Determination of Salicylic Acid and Its Metabolites in Urine
Authors: Farid Nagy A , Born Gordon S , Kessler Wayne V , Shaw S M , Lange W E
Published: January 2020

We describe an improvement in the Levy and Procknal method [J. Pharm. Sci. 57, 1330 (1968)] for determination of salicylic acid and its metabolites in urine. Salicylic acid and salicyluric acid are successively extracted from 1 or 2 ml of urine (acidified with HCl) by two 10-ml portions each of carbon tetrachloride and ethylene dichloride. The extracts of each solvent are shaken with 5 ml of ferric nitrate solution (a 10-fold dilution of 17 g of Fe(NO3)3·9H2O in 1 liter of 70 mmol/liter HNO3). The aqueous phases are centrifuged and their absorbances measured at 530 nm. For total salicylate, 3 ml of urine and 3 ml of HCl are heated in a partially evacuated serum vial at 100 °C for 16 h and then salicylic acid is assayed in the hydrolyzed sample. Recovery of a weighed oral dose of sodium salicylate in urine was 105.4%; it was 127.9% by the Levy and Procknal method for the same sample. The improved method is faster and more accurate.
Automated Elution Electrophoresis: A Potential Clinical Tool
Authors: Scott Charles D , Lee Norman E , Perkins Arthur W
Published: January 2020

An elution electrophoresis system in which a porous packed bed is used for separation and a flow photometer or colorimeter for continuous monitoring of the eluate may be capable of rapid, high-resolution analysis of serum proteins and other protein mixtures with very little manual labor. In a prototype of such a system we used a cooled separations column, 3 mm in diameter and 40 cm long, containing polyacrylamide beads. Samples are introduced, via a microsyringe, through a septum at the column midpoint. Typical analyses for serum proteins or serum isoenzymes require an electrophoresis time of about 30 min at 1200 V.
Development of a Multipurpose Optical System for Use with a Centrifugal Fast Analyzer
Authors: Burtis C A , Bostick W D , Johnson W F
Published: January 2020

A Centrifugal Fast Analyzer is basically a sophisticated photometric measuring device containing a multicuvet rotor as its major component. Several reactions are simultaneously initiated in the rotor, which is then rotated through a stationary optical monitor and the resulting signals acquired and processed by an on-line data system. With the miniature version of this analyzer, one has the option of directing the incident optical beam, via a fiber optical bundle, into the cuvets of the spinning rotor in either a 90° or a 180° orientation relative to the analyzer's photodetector. The combination of newly designed rotors and a flexible optical system having multiple configurations has provided a versatile system in which one can measure the transmittance, fluorescence, chemiluminescence, or light-scattering (either turbidimetrically or nephelometrically) properties of the ensuing reaction species, all with a single analyzer. This flexibility in choosing the optical mode in which a particular set of reactions is to be monitored provides the analyst with a powerful and versatile analytical tool for developing new methods for use in various clinical laboratory applications, including chemistry, toxicology, immunology, and hematology.
Application of Differential Light Scattering to the Latex Agglutination Assay for Rheumatoid Factor
Authors: Blume Philip , Greenberg Leonard J
Published: January 2020

When a suspension of particles is irradiated with a collimated beam of monochromatic polarized light of a wavelength close to the particle size and the intensity of the scattered light is measured as a function of angle, the scatter intensity is characterized by a series of relative maxima and minima. The nature of the signal depends on several significant variables that are characteristic of the particles. We have constructed a differential light scatter photometer and have applied the technique to analysis of rheumatoid factor by using latex particles coated with fraction II gamma-globulin. The results suggest that such a photometer may have potential applications in antigen-antibody assays based on the use of sensitized particles.
Potential for Clinical Use of the Analytical Laser Microprobe for Element Measurement
Authors: Glick David , Marich Kenneth W
Published: January 2020

Use of the laser microprobe for rapid emission spectroscopic analysis of elements in microscopic samples of biological material is described. The technique depends on vaporization of the microsample with a focused laser beam at a temperature that renders the vapor incandescent for spectrochemical analysis. Spectral line intensities are recorded photographically with densitometry of the negatives or photoelectrically. Current capability permits analysis of about 10-8 to 10-10 g of tissue, with detection limits of 10-12 to 10-15 g of element. Groups of elements can be simultaneously analyzed. Minimum sample preparation is required, and the analysis is done on the stage of a light microscope, usually on an air-dried sample on a plastic slide. We exemplify the technique in analysis of gold in cultured fibroblasts treated with gold salts and in human skin after treatment with gold salts for rheumatoid arthritis, in element changes in biopsies of transplanted human hearts, and in unique profiles of groups of elements in human cancer tissue.
Making Enzymatic Methods Optimum for Measuring Compounds with a Kinetic Analyzer
Authors: Atwood John G , DiCesare Joseph L
Published: January 2020

Kinetic enzymatic methods for analysis of substrates can be made optimum for a sensitive photometric analyzer by adjusting the activity of the triggering (catalyzing) enzyme so that the reaction rate is maximum at the time of measurement. At this optimum activity, the exponential time constant for exhaustion of substrate equals the time between triggering and rate measurement. The scale factor (defined as measured activity divided by sample concentration in the reaction mixture) is the same for all tests. Sensitivity to substrate concentration is predictable from instrumental absorbance uncertainty and molar absorptivity of the absorbing species. These predictions from Michaelis theory were verified experimentally for pyruvate and lactate triggered with lactate dehydrogenase, for glucose triggered with hexokinase, and for triglycerides triggered with glycerol kinase, the reaction rate being measured 30 s after triggering. Sensitivities of 1.5 x 10-7 mol/ liter were achieved. Serum diluted 1000-fold and analyzed for glucose gave a repeatability of 25 mg/liter with linearity to 4.0 g/liter. Samples diluted 300-fold and analyzed for triglycerides gave 30 mg/liter repeatability, with linearity to concentrations exceeding 3.0 g/liter.
Application of Electron Spin Resonance to Determination of Serum Drug Concentrations
Authors: Montgomery Mark R , Holtzman Jordan L , Leute Richard K
Published: January 2020

Displacement of spin-labeled drugs from drug-specific antibodies in human serum can be measured by use of electron spin resonance. This "spin immunoassay" has advantages of speed, simplicity, and small volume requirements over other standard techniques such as gas—liquid, liquid—liquid, or thin-layer chromatography. Unlike radioimmunoassay, there is no need to separate free and bound label and there are no radiation hazards. We present clinical and experimental data showing how this method can be applied to the routine determination of morphine and diphenylhydantoin concentrations in serum.
Convenient method for checking detector response of spectrophotometers at three wavelengths.
Authors: Frings C S , Muscat V I , Waldrop N T
Published: January 2020

We describe the use of a water-soluble dye, available as green food coloring, to check the linearity of detector response of spectrometers and spectrophotometers at three wavelengths. The dye has absorbance maxima at 257 nm, 410 nm, and 630 nm. The advantages of using this solution for this important instrument function check are: one solution with absorption maxima in the opposite ends of the visible spectrum as well as in the ultraviolet region; safe; inexpensive; readily available; easy to use; absorbance not dependent on pH from 2.5 to 10.0 or on temperature from 4 to 56 degrees C.
A computerized system for storage and statistical analysis of clinical amino acid data.
Authors: Schoengold D M , Miner D J , deFiore R H , Hamilton P B
Published: January 2020

We have developed a system of computer programs to expedite analyses of amino acid data obtained in a clinical environment. The system contains a program for building and maintaining libraries of chromatogram data, and a program for retrieval of data on the basis of any of its associated biographical characteristics. Several programs have been written that work with data drawn from the libraries. They provide for easy presentation, manipulation, or statistical analysis of the data. Included are comparison of population means and variances and intra-population correlation analyses. A minicomputer that has 12K of core storage is adequate for use with the system.
Effect of time and temperature on blood P50 and 2,3-diphosphoglycerate measurements.
Authors: Hellerstein S , Bunthrarungroj T
Published: January 2020

The oxygen affinity of hemoglobin is affected by various factors, including blood pH and erythrocyte 2,3-diphosphoglycerate. The latter and blood P50 values are unchanged when aliquots of blood from normal individuals are permitted to stand at room temperature for as long as 7 h. Our data also show that equilibration in a 37 degrees C tonometer for times ranging from 0.5 to 1.5 h causes no detectable alterations in P50 values.
Electrophoretic amylase fractionation as an aid in diagnosis of pancreatic disease.
Authors: Legaz M E , Kenny M A
Published: January 2020

Six alpha-amylase (EC 3.2.1.1) isoenzymes have been resolved electrophoretically on cellulose acetate membranes in a discontinuous buffer system. The fastest migrating isoenzymes are of salivary origin (S1, S2, S3), the slower ones of pancreatic origin (P1, P2, P3). We determined the amylase isoenzyme distribution in the sera of 240 subjects. A specific pancreatic isoenzyme (P3) was observed in all clinically diagnosed cases of acute or chronic pancreatitis as well as in 15 of 40 renal-transplant patients. Moreover, P3 isoenzyme activity declined during apparent recovery from pancreatitis. The P2 isoenzyme appeared in 95% of all specimens, P1 in only 2%. The pancreatic isoenzymes were preferentially excreted in the urine of both renal-transplant patients and normal individuals. The major salivary isoenzyme, S1, was observed in 95% of all serum and urine samples; however, the S2 and S3 appeared less consistently. Our method is simple and rapid, and quite applicable for use in clinical evaluation of patients with pancreatitis or with certain nonpancreatic dysfunctions.
Thermodynamic aspects of some radioassays.
Authors: Keane P M , Walker W H , Gauldie J , Abraham G E
Published: January 2020

In a number of radioimmunoassays and radiotransin assays, effective equilibrium constants have been measured at different temperatures in order to define the relative contribution of changes of entropy and enthalpy to the change in free binding energy. In systems with a large enthalpy component, the lowest possible incubation temperature maximizes sensitivity, and control of temperature throughout the assay is important. Conversely, when enthalpy change is small, a high temperature allows rapid attainment of equilibrium without loss of sensitivity. At a theoretical level, the thermodynamic characteristics of binding may allow some insight into the nature of the binding process.
Determination of phenprocoumon, an anticoagulant, in human plasma.
Authors: DeWolf F A , VanKempen G M
Published: January 2020

Phenprocoumon is extracted from acidified plasma, the organic phase evaporated, and part of the residue, in ethanol, is quantitatively applied to a thin-layer plate. After separation, the quantity of phenprocoumon is assayed by fluorescence densitometry in situ. Results are reproducible to about 2.5%. The lower limit of detection is 0.1 mg/liter, which makes the method fully applicable to human plasma, because therapeutic concentrations range from 1 to 3 mg/liter. Seven determinations can be made within 3 h. For toxicological purposes, a qualitative analysis can be done in a shorter time, because the phenprocoumon spots are visible under ultraviolet light at 254 nm.
Kinetic determination of hexosaminidases for Tay-Sachs heterozygote screening.
Authors: Zinterhofer L , Schuttringer G
Published: January 2020

We describe a method for continuous kinetic measurement of hexosaminidase activity, and have applied it to detection of heterozygotes for Tay-Sachs gene. In contrast to existing single-point methods, a ph of 4.5, which is optimal for hexosaminidase activity on the substrate (4-methylumbelliferyl-N-acetyl-beta-d-glucosaminide) is maintained while the increase in fluorescence produced by 4-methylumbelliferone, the reaction product, is being determined. Under the conditions described the reaction follows zero-order kinetics, and activity is linearly related to serum dilution. There is a fairly narrow but practicable range of optimal substrate concentration. The presence of substrate in concentrations exceeding 1 mmol/liter results in progressive quenching of fluorescence and a decrease in apparent enzyme activity. The procedure is standardized with 4-methylumbelliferone in a solution of the substrate. This method is adaptable to use with automated multiple-point discrete-sample devices and especially with the fluorometric kinetic analyzers now being developed.
Factors contributing to intra-individual variation of serum constituents: Physiological day-to-day variation in concentrations of 10 specific proteins in sera of healthy subjects.
Authors: Statland B E , Winkel P , Killingsworth L M
Published: January 2020

Using an automated immunoprecipitin method, we assayed human sera for 10 proteins: haptoglobin, orosomucoid, transferrin, alpha1 antitrypsin, alpha2-macroglobulin, IgG, IGa, IgM, complement C3, and complement C4. Blood from 14 healthy subjects (25-40y) was sampled on six separate days. From each venipuncture serum was divided into four eliquots; two were assayed on the day of venipuncture and two were frozen and kept until the end of the study, when all of the frozen samples were analyzed in one batch. With this experimental design, batch-to-batch analytical variation could be estimated, and we avoided confounding it with the biological variation. Data analysis was based on the analysis of variance technique. The average physiological intra-individual coefficient of variation ranged from 2.5% for transferrin to 11.1% for orosomucoid. THe interindividual variation ranged from 9.5% for transferrin to 70.5% for haptoglobin and the ratio between intra-individual variation and interindividual variation ranged from 0.66 for IgM to 0.26 for orosomucoid and transferrin.
Automated radioimmunoassay of choriomammotropin (human placental lactogen).
Authors: Castro A , Prieto I , Wunsch C , Ertingshausen G , Malkus H
Published: January 2020

We describe a totally automated procedure for radioimmunoassay of choriomammotropin, in which all phases of the assay are automated in a single system ("Centria"). This system permits the simultaneous incubation and separation of many samples in a nonequilibrium assay, and measurements are obtained in less than 30 min. Results for clinical samples by reference radioimmunossay methodology and with the Centria system compared uniformly well: y=0.91x - 0.87;r=0.94. The coefficient of variation for samples run in duplicate on the same day was 5.2%, 7.4% for samples run on different days. The specificity, sensitivity, simplicity, and speed of this system makes it a useful new tool for kinetic, nonequilibrium immunoassay.
Simple modifications of three routine in vitro tests of thyroid function.
Authors: Pain R W
Published: January 2020

Semi-automation of equipment and simple modifications of technique reduced the work load without loss of diagnostic accuracy for three commonly used in vitro tests of thyroid function (total thyroxine, thyrobinding index, and free thyroxine index). Major innovations were the use of serum standards for all tests and having each duplicate for tests performed by a different technician. Attention is drawn to the false-positive and false-negative errors that occur when the 95% euthyroid limits is the sole reference range used.
Modified determination of total and inorganic mercury in urine by cold vapor atomic absorption sectrometry.
Authors: Littlejohn D , Fell G S , Ottaway J M
Published: January 2020

In this procedure a single-beam spectrophotometer is used without background correction. By the method of Magos [Analyst (London) 96, 847 (1971)] mercury in undigested urine is complexed to L-cysteine in acid solution. At high pH and in the presence of stannous ions, mercury ions are reduced to elemental mercury. The mercury vapor is partitioned above the reagent solution in a specially designed chemical reduction apparatus similar in principle to that used by Kubasik et al. [Clin. Chem. 18, 1326 (1972)]. The vapor is then flushed by air through an "absorption" cell, where the absorption of the mercury line at 253.7 nm is measured. The value obtained for inorganic mercury subtracted from that for total mercury gives a value for organic mercury. CV's for the inorganic mercury procedure at 40 and 5 mug/liter concentrations were 3.1% and 7.5%, respectively. The detection limit is 0.82 mug/liter. The CV for the total-mercury procedure (20 mug/liter) was 2.6%. Mean analytical recoveries of organic and inorganic mercury were 96.5% and 101%, respectively. We investigated storage conditions for urine and compared results by the present technique with those by activation analysis. Our method is a convenient way to screen individuals who have been exposed to a mercury hazard.