bluuebunny/crossref_metadata_2025 huggingface dataset:
A radioimmunoassay for human serum myoglobin: method development and normal values.
Authors: Rosano T G , Kenny M A
Published: January 2020

We describe a radioimmunoassay that will measure both normal and above-normal concentrations of myoglobin in serum. Myoglobin isolated from human pectoralis muscle was purified by (NH4)2SO4 fractionation and Sephadex gel filtration and injected into rabbits to elicit antisera. Myoglobin was radiolabeled by an acylation with [125]-3-(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester. With the purified myoglobin and antisera, we then developed a radioimmunoassy that involves simultaneous regent addition, a 3.5-h incubation at 37 degrees C, and separation of the antibody-bound fraction by precipitation with polyethylene glycol. Information is given on detection limit, precision, linearity, recovery, and specimen preservation. Cross-reactivity to human hemoglobin is negligible. Finally, we investigated the possible relationship between serum myoglobin concentration and muscle mass.
Creatine kinase and its isoenzymes in the serum of women during pregnancy and the peripartum period.
Authors: McNeely M D , Baerris B , Papsin F R , Lyons E , Schipper H
Published: January 2020

In serum obtained from 28 women before, during, and after normal labor and delivery, creatine kinase activity was seen to be distinctly elevated immediately after labor and 24 h later, but had returned to normal six weeks later. In most cases the increase was due to the MM isoenzyme and was attributed to skeletal-muscle damage associated with labor. In 15 cases, the BB isoenzyme was observed, and in three patients the MB isoenzyme. Cord blood, which contains all three isoenzymes, may be the source of the MB and BB isoenzymes. Uterine muscle contains exclusively BB isoenzyme, and therefore uterine muscle damage is a likely source of the serum BB isoenzyme. The relationship among these isoenzymes in serum after delivery must be recognized, to avoid misdiagnosing myocardial infarction at this critical time.
Adenosine deaminase activity in lymphocytes of normal persons, leukemic patients, and kidney-transplant recipients.
Authors: Yasmineh W G , Brynes R K , Lum C T , Abbasnezhad M
Published: January 2020

We determined the activity of adenosine deaminase in normal lymphocytes from peripheral blood, leukemic cells from patients with various hematologic malignancies, and mononuclear cells from kidney-transplant patients, by adapting the method of Hopkinson et al. [Ann. Hum. Genet. 32, 361 (1969)] to the centrifugal analyzer. Normal lymphocytes separated on Ficoll-Hypaque from peripheral blood had a mean activity of 1.42 U/10(9) cells (SD = 0.57; N = 33). Cells similarly isolated from patients with acute lymphoproliferative and myeloproliferative disease generally showed greater values, but the ranges overlapped. Cells from patients with chronic lymphocytic leukemia had activities that were within or below the normal range. Mononuclear cells isolated from the peripheral blood of 15 stable renal allograft recipients showed a mean activity of 1.90 U/10(9) cells (SD = 0.59), not significantly different from normal. In contrast, cells from five patients with biopsy-proven allograft rejection showed a mean activity of 12.84 U/10(9) cells (SD = 10.10), which was significantly different from that of the stable patients.
Electrophoretic patterns for serum glycoproteins reflect the presence of human breast cancer.
Authors: Silverman L M , Dermer G B , Tökes Z A
Published: January 2020

We describe a group of glycoproteins that are synthesized and released by human breast tumors maintained in organ culture and similar glycoproteins released by a human breast carcinoma cell line (BT-20). The electrophoretic mobility of these glycoproteins on cellulose acetate is consistent with increased glycoprotein-staining material present in the alpha2- to beta-globulin region of serum glycoprotein electropherograms from patients with breast cancer. Moreover, after mastectomy, this glycoprotein material in serum decreases to concentrations seen in a control population of patients with benign breast lesions. Patients with proven metastatic breast cancer have patterns reflecting their clinical status: those who respond to treatment have glycoprotein electropherograms similar to the group of patients with benign breast lesions, while those who do not have increased amounts of alpha2- beta-glycoprotein. We believe serum glycoprotein measurements in breast-cancer patients reflect the presence of glycoproteins that are released by the malignant cells and enter the circulation.
Use of protein containing magnetic microparticles in radioassays.
Authors: Ithakissios D S , Kubiatowicz D O
Published: January 2020

We describe a radioassay method that involves the use of magnetic protein microparticles composed of a water-insoluble protein matrix containing magnetically responsive material. We define two different types of particles according to the mechanism of action: (a) The substrate is sorbed nonspecifically by the protein matrix of the particle or by a second substance such as charcoal or ion-exchange resin incorporated within the protein matrix of the particle. These particles are useful for separating free from bound substrate. Examples of these are albumin magnetic microparticles for use in a total thyroxine radioassay and triiodothyronine uptake test, or albumin magnetic microparticles containing charcoal for use in a vitamin B12 radioassay. (b) The substrate is sorbed specifically by a binding protein incorporated within the matrix of the particles. The binding protein can include antibodies or other specific nonimmune proteins. Particles of this type are useful in solid-phase radioassays. These particles are exemplified by albumin magnetic microparticles containing sockeye salmon serum, used in a solid-phase B12 radioassay. We discuss the methods for the preparation of both types of magnetic microparticles and their use in radioassays. We describe a unique inexpensive magnetic separation rack, which provides simple, fast, and reproducible separation of the magnetic microparticles from their suspending medium during the assay.
Radioimmunoassay of lysergic acid diethylamide (LSD) in serum and urine by using antisera of different specificities.
Authors: Ratcliffe W A , Fletcher S M , Moffat A C , Ratcliffe J G , Harland W A , Levitt T E
Published: January 2020

We raised high-titre antisera to two LSD-bovine serum albumin conjugates, one linked via the indole nitrogen, the other via the amide side-chain. The antisera were specific for different parts of the LSD molecule, as demonstrated by cross-reactivity studies with LSD, its metabolites, ergot alkoloids, and closely related compounds. The antisera were used to develop a double-antibody radioimmunoassay with a detection limit of about 0.4 mug of LSD per liter of unextracted urine or serum. We saw no nonspecific interference by urine, serum, or from a series of commonly used drugs. There was good correlation between immunoassay values obtained with the two antisera (r = 0.91). However, the antiserum linked via the indole nitrogen gave consistently higher results for samples from persons who had taken LSD, owing to greater cross-reactivity with LSD metabolites. Radioimmunoassay by use of two such antisera is a more specific screening procedure for LSD abuse than has been available previously. In addition, antisera cross-reacting with LSD metabolites allow measurement of these compounds, for which there is no satisfactory method at the concentrations found in biological fluids in man.
Copper, zinc, magnesium, and calcium in plasma and cerebrospinal fluid of patients with neurological diseases.
Authors: Bogden J D , Troiano R A , Joselow M M
Published: January 2020

We investigated whether information on concentrations of some trace-mental concentrations in blood plasma or cerebrospinal fluid, or both, could be of value in diagnosis or management of various neurological diseases, and whether concentrations in plasma could serve as a means of estimating the protein or metal concentrations in cerebropsinal fluid. Samples of both from 82 patients were analyzed for copper, zinc, magnesium, and calcium by atomic absorption spectrophotometry. Protein concentrations in cerebrospinal fluid were also determined. Metal and protein concentrations in plasma and in cerebrospinal fluid were not strongly enough correlated to permit the estimation of one from the other. However, the correlation coefficients between calcium in plasma and cerebrospinal fluid (r = 0.41), magnesium and protein in cerebrospinal fluid (r = 0.40), magnesium in plasma and calcium in cerebrospinal fluid (r = 0.36), and magnesium and calcium in cerebrospinal fluid (r = 0.66) were statistically significant (P less than .01). Patients with cerebral infarctions had abnormally high copper concentrations in their plasma and cerebrospinal fluid. The ratio of plasma copper to plasma zinc was also significantly higher in cases of cerebral infarction.
Radioimmunoassay for adenosine 3', 5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate in human blood, urine, and cerebrospinal fluid.
Authors: Goldberg M L
Published: January 2020

I describe a highly sensitive and very specific assay for cyclic AMP and cyclic GMP in body fluids. An adaptation of the method of Harper and Brooker [j. cyclic Nucleotide Res. 1,207 (1975)], its advantage lies in the fact that the simple acetylation of the samples--a procedure developed by these workers--greatly increases specificity and sensitivity beyond those of previous methods and permits the measurement of femtomole quantities of both cyclic nucleotides without prepurification. Because the range of the cyclic AMP assay is 20-200 fmol, and that of cyclic GMP 10-15 fmol, experiments can be performed with use of only microliters of fluid. The method requires no extraction and thus no complicated corrections are necessary for analytical recovery.
Genetic Variability of Clinical Chemical Values
Authors: Havfik Richard , Garrison Robert , Fabsitz Richard , Feinleib Manning
Published: January 2020

A study of cardiovascular risk factors in middle-aged twin men provided an opportunity to test for genetic variability in the SMA 12/60 (Technicon) battery of clinical chemistry tests. Classical twin methodology was used to analyze the variation of monozygotic and dizygotic twins. In addition, frequency of co-twin contact was used to control for ef¬fects of differences in shared environment. Genetic vari¬ability played a definite role in controlling four of the 11 reported tests: one-hour serum glucose, serum urea ni¬trogen, uric acid, and bilirubin. No genetic variation was found for lactate dehydrogenase, phosphorus, and alkaline phosphatase. Significantly higher means for calcium, total protein, albumin, and aspartate aminotransferase in monozygotic twins precluded any statement about heredity and environment for these tests.
New substrate for fluorometric determination of gamma-glutamyltransferase activity in serum.
Authors: MacQueen J D , Driscoll R C , Gargiulo R J
Published: January 2020

We describe the preparation of a new substrate, N-gamma-L-glutamyl-5-aminoisophthalic acid dimethyl ester hydro-chloride, for the fluorometric determination of gamma-glutamyltransferase activity in serum by the "front-surface" technique. Details of the resulting method are provided. The final reaction mixture contains 4 mmol of the substrate per liter of tris(hydroxymethyl)aminomethane (100 mmol/liter) and glycylglycine (75 mmol/liter) at pH 8.2 (25 degrees C).
Choice of monitoring isotope in double label radioimmunoassays with nonimmunological separation techniques.
Authors: Peeters T L , Vantrappen G R
Published: January 2020

We discuss the suitability of some radioactive isotopes as volume markers in radioimmunoassays, from a radiochemical point of view. For three eligible isotopes (22Na, 60Co, and 75Se) we studied the concentration of the marker in the precipitate formed in the separation phase of radioimmunoassays. For all those kinds of separations tested (charcoal, ammonium sulfate, polyethylene glycol, and ethanol), binding or coprecipitation was virtually absent or negligible with 22Na but 75Se was strongly concentrated in the precipitate. Concentration of 60Co occurred only with charcoal and ethanol precipitation. Because heavy metals tend to bind to serum proteins, we conclude that of all radioactive isotopes commercially available only 22Na should be used in radioimmunoassays with nonimmunological separation methods.
Preparation of a phospholipase C-antihuman IgG conjugate, and inhibition of its enzymatic activity by human IgG.
Authors: Wei R , Riebe S
Published: January 2020

We labeled IgG with phospholipase C, using 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide. Enzymaticactivity of the resulting conjugate was inhibited when it was complexed with human IgG, but rabbit or goat IgG was not effective in suppressing the enzyme activity. Normal erythrocytes were used as substrate for the enzyme, enzymatic activity being assessed by measuring the release of hemoglobin. The substrates for phospholipase C are phospholipids, which are major components of the erythrocyte membranes. Hence, the phospholipids in the membranes are viewed as being "immobilized." Perhpas such immobilization of substrate may be a requisite to the inhibition phenomenon.
Lactate dehydrogenase isoenzymes linked to beta-lipoproteins and immunoglobulin A.
Authors: Trocha P J
Published: January 2020

A clinically asymptomatic individual had an abnormal electrophoretic pattern for lactate dehydrogenase, seven isoenzyme bands being present. This atypical pattern was a result of an interaction of lactate dehydrogenase with immunoglobulin A and beta-lipoprotein. Lactate dehydrogenase enzymes LD2 and LD3 were the isoenzymes responsible for the formation of the complexes.
Direct micromethod for colorimetry of serum ornithine carbamoyltransferase activity, with use of a linear standard curve.
Authors: Vassef A A
Published: January 2020

Determination of serum ornithine carbamoyltransferase (EC 2.1.3.3) activity can be a valuable diagnostic tool in the detection of liver diseases involving cytolytic processes. I describe a micromethod for measuring this activity in serum, in which the reaction product, citrulline, is measured colorimetrically in the incubation mixture without prior deproteinization. To eliminate the interference of serum protein precipitation, the concentration of sulfuric acid in the color reagent has been decreased, without substantial loss of sensitivity. Optimizing the conditions of citrulline determination, in which antipyrine and 2,3-butanedione monoxime are used, has resulted in a linear standard curve. The color formed by citrulline is found to be stable in room lighting and sensitive only to direct sunlight. The precision of the method is inversely correlated to serum enzyme activity, the CV varying between 4.6 and 21.1%.
Relative merits of two electrophoretic and two column-chromatographic kets for determining serum creatine kinase isoenzyme MB activity.
Authors: Hamlin C , Ackerman E
Published: January 2020

Prospective analysis (by use of commercially available kits) of 339 validated admission specimens from patients consecutively admitted to a cardiac monitoring unit revealed sensitivities of 68 and 63% for two electrophoretic assays (Bioware and Helena) and up to 73 and 77% for two chromatographic assays (Roche and Worthington). Specificities were 94.8, 96,6, 90.2, and 89.7% and efficiencies were 90.2, 90.7, 87.0, and 87.0, respectively. Although these results differ from early reports, they agree well with several recent studies. However, 86 (25%) of the specimens gave at least one discrepant result based on clinical evaluation of the patients, and 68 (20%) gave at least one discrepant result between methods.
Results with commercial radioassay kits compared with microbiological assay of folate in serum and whole-blood.
Authors: McGown E L , Lewis C M , Dong M H , Sauberlich H E
Published: January 2020

We compared results with three commercial folate radioassay kits [Bio-Rad, New England Nuclear (NEN), and RIA Products] with those by microbiological assay for more than 200 samples of human serum and whole blood. All but one kit (NEN) compared favorably with the microbiological assay for serum samples, although there were notable diagnostic discrepancies. Two kits (NEN and Bio-Rad) were tested on whole-blood samples; both yielded values significantly higher than those by microbiological assay. The frequency distributions of erythrocyte folate data differed strikingly between the two kits; the NEN method yielded a much narrower range of normal values than did either the Bio-Rad or the microbiological assay. Radioassay kits appear to be suitable diagnostic agents for serum folate, if the behavior of a particular kit is investigated thoroughly before its routine use. However, the diagnostic value of radioassays of erythrocyte folate needs to be validated.
Kinetic centrifugal analyzer and manual determination of serum urea nitrogen, with use of o-phthaldialdehyde reagent.
Authors: Levinson S S
Published: January 2020

o-Phthaldialdehdye reagent was used in assaying urea with a miniature centrifugal analyzer. Seventeen samples can be assayed kinetically in 2 min at a current cost of less than a penny per test. The reaction is initiated in the instrument upon simultaneous mixing of the o-phthaldialdehyde reagent containing 10 microliter of sample with dilute sulfuric acid/N-(1-naphthyl)ethylenediamine dihydrochloride reagent. The reaction is monitored kinetically at 546 nm during the initial 2 min, when the rate is nearly constant. The reaction of o-phthaldialdehyde with urea reaches equilibrium by about 7 or 8 min, but the method cannot be used for end-point analysis because of interference by the blank reaction. A 1.2 g/liter solution of o-phthaldialdehyde used in conjunction with 2 mol/liter sulfuric acid solution was found to be most suitable for centrifugal analyzers. I also describe a manual version of the procedure that gives a linear standard curve up to 1.0 g of urea nitrogen per liter.
Hyperlipoproteinemia phenotype determination by agarose gel electrophoresis updated.
Authors: Papadopoulos N M
Published: January 2020

Hyperlipoproteinemia phenotyping has been re-evaluated by an improved agarose gel electrophoretic test. Eight varieties of serum lipoprotein electrophoretic patterns are demonstrated, two from clinically normal normolipidemic subjects and six from patients with hyperlipoproteinemia that include the phenotypes 1, 2a, 2b, 3, 4, and 5. The sharp delineation of the lipoprotein patterns achieved by this technique facilitates the differentiation and identifcation of the lipoprotein phenotypes. Furthermore, the lipoprotein patterns can be visually evaluated, correlated with their corresponding serum lipid values, and associated with the clinical and hereditary features of the hyperlipoproteinemia phenotypes. This test system can also be used in clinical trials and epidemiological studies.
Faster determination of clottable fibrinogen in human plasma: an improved method and kinetic study.
Authors: Inada Y , Okamoto H , Kanai S , Tamaura Y
Published: January 2020

Clottable fibrinogen in human plasma was determined by measuring spectrophotometrically the increase in turbidity with time due to the fibrinogen-fibrin conversion with thrombin. From the maximal absorbance, Amax, at 450 nm obtained 2 min or less after thrombin as added to plasma, we estimated the fibrinogen concentration in plasma of normal subjects and patients. Analysis of the rate of the absorbance increase yielded the Km value, 1.6 X 10(-5) mol/liter, which closely agrees with the Km of 1.2 X 10(-5) mol/liter obtained by analysis of the fibrinopeptides released from fibrinogen.
Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30, and 37 degrees C.
Authors: Buhl S N , Jackson K Y
Published: January 2020

We report optimal conditions for assaying highly purified human lactate dehydrogenase isoenzymes with the lactate-to-pyruvate and pyruvate-to-lactate reactions, as they apply to human serum. Interconversion of results between reactions is not practicable. Measurements of lactate dehydrogenase in either reaction direction at 25, 30, or 37 degrees C can be equally reliable if the volume fraction and the resulting deltaA/min is small. However, for interinstrument and interlaboratory comparisons, results from the lactate-to-pyruvate reaction are more reliable.
Comparison of product moment and rank correlation coefficients in the assessment of laboratory method-comparison data.
Authors: Cornbleet P J , Shea M C
Published: January 2020

We have studied the effects of range and distribution of data on product moment and rank correlation coefficients when deviation from a linear relationship was due solely to experimentally produced random error. All correlation coefficients (Pearson r, Spearman rho, and Kendall tau) were markedly influenced by the range of the data, and, for the rank correlation coefficients, the effect of range varied for different data distributions. While correlation coefficients may be useful in assessing whether an association exists between two variables, they are not useful in assessing the degree of random error about the regression line when a strong linear association is presumed to exist between the two variables. Thus, neither product moment nor rank correlation coefficients are of value in analysis of laboraoty method-comparison data. The standard deviation of the residual error of regression should be calculated as a measure of the random error about the regression line.
A micro-scale method for liquid scintillation counting, applicable to radioimmunoassay of steroids and substances of comparable relative molecular mass.
Authors: Grotjan H E , Steinberger E
Published: January 2020

A major expense of radioimmunoassay involving tritiated tracers is that of liquid scintillation counting. We present a micro-scale method that markedly reduces the cost of liquid scintillation counting. The radioimmunoassay is done in 10 X 50 mm tubes. The antibody-bound tracer is precipitated with a second antibody, the precipitate is resuspended in 0.1 ml of water, and 0.3 ml of dioxane is added. One milliliter of a toluene-based scintillation cocktail is added, and the tube is capped and placed in an adapter for liquid scintilation counting. When this method was applied to a double-antibody testosterone radioimmunoassay, it performed comparably to assays counted by the usual method.
Multilayer film elements for clinical analysis: applications to representative chemical determinations.
Authors: Spayd R W , Bruschi B , Burdick B A , Dappen G M , Eikenberry J N , Esders T W , Figueras J , Goodhue C T , LaRossa D D , Nelson R W , Rand R N , Wu T W
Published: January 2020

Using the general concept of a dry multilayer analytical element, we can change chemical procedures and configurations to assay several blood components. In the assay of serum urea nitrogen, urease in the reagent layer catalyzes the hydrolysis of urea. A semipermeable membrane excludes aqueous base, but allows ammonia to diffuse to an underlying indicator layer. For the amylase determination, the enzyme hydrolyzes a dyed-starch substrate coated on top of the spreading layer; this produces small fragments, which diffuse to a registration layer. The increase of absorbance at 540 nm is correlated with amylase activity. Bilirubin complexes with a cationic polymer at the interface between the spreading and reagent layers. The direct reading at 460 nm allows determination of total bilirubin in the range 1 to 500 mg/liter. Tirglycerides are hydrolyzed in the spreading layer, and the resulting soluble glycerol readily diffuses into the reagent layer, where it is phosphorylated and subsequently oxidized by glycerophosphate oxidase to yield dihydroxyacetone phosphate and hydrogen peroxide. Peroxidase catalyzes production of a color commensurate with the hydrogen peroxide produced.
Coupled reactions of immobilized enzymes and immobilized substrates: clinical application as exemplified by amylase assay.
Authors: Barabino R C , Gray D N , Keyes M H
Published: January 2020

We described a partitioned enzyme-sensor system, which incorporates an immoblized substrate and three or more discrete immobilized enzymes. This instrument measures alpha-amylase activity by passing the solution containing alpha-amylase over a column packed with immobilized starch. The resulting oligosaccharides are successively exposed to a column or columns containing immobolized glucose oxidase, catalase, glucoamylase or maltase, and glucose oxidase. The resulting hydrogen peroxide is detected by a three-electrode amperometric cell. All immobilized reagents were immobilized on a particulate, porous alumina to allow rapid and constant flow rate. With use of less than optimum immobilized reagents, alpha-amylase activity has been measured from about 5 to 200 kU/liter with a 50 microliter sample size. Lack of sensitivity is predominantly attributable to the low activity and low stability of immobilized maltase and glucoamylase. We believe that a clinical test using this system is feasible and desirable because the immobilized reagent system should allow for testing of alpha-amylase with excellent precision, convenience to the operator, and low cost.
External quality-control survey of cholesterol analyses performed by 12 lipid research clinics.
Authors: Lippel K , Ahmed S , Albers J J , Bachorik P , Muesing R , Winn C
Published: January 2020

We report accuracy and precision achieved in the automated analysis for cholesterol in a long-term multilaboratory study, presenting and evaluating the significance of data accumulated by 12 Lipid Research Clinics (LRC's) in the analysis of 18 unknown surveillance pools during three years. The average bias for all pools and for 13 autoAnalyzer II (Technicon Instruments Corp., Tarrytown, N.Y. 10591) instruments in the 12 clinics was -0.41% (range -1.2 to +0.3%), as compared to values established by reference methodology. The regression equation relating observed cholesterol values (y) to reference values (x) was: y = 0.35 + 0.977x. The bias varied from pool to pool (-2.3 to +5.3%), positive biases being observed for pools with cholesterol concentrations less than 1.4 g/liter, and negative biases for those pools with higher concentrations. Total standard deviations ranged between 25 and 75 mg/liter, and total CV's for most individual instruments were between 1 and 3%. Of the variability for a particular pool, less than 20% was due to differences among instruments, and within- and between-run variabilities were approximately equal. These trends were the same as those previously observed [Clin. Chem. 23, 1744 (1977)] in the analysis of bench control pools of known cholesterol concentration.
Liquid-chromatographic determination of serotonin in serum and plasma.
Authors: Sasa S , Blank C L , Wenke D C , Sczupak C A
Published: January 2020

Using high-performance liquid chromatography with electrochemical detection, we determined serotonin in plasma from parkinsonian patients being treated with L-3,4-dihydroxyphenylalanine or N-(DL-seryl)-N'(2,3,4-trihydroxybenzyl)hydrochloride plus L-3,4-dihydroxyphenylalanine ("Sinemet") and in serum from a blood bank, from "normal" persons, and a pooled specimen from a hospital clinical laboratory. The values obtained for the two groups of Parkinson's disease patients showed no significant difference. Long-term storage on solid CO2 was xhown to be an adequate technique for preserving samples. The mean (+/-SEAM) normal value obtained for serotonin in serum was 146 +/- 46 microgram/liter (n = 23), a result in harmony with that previously obtained [Clin. Chem. 20, 812 (1974)] by fluorometry. In comparison to other methods for measurement of serotonin in serum or plasma, we believe that the present scheme offers greater selectivity, sensitivity, and precision.
An improved technique for immunofixation of electrophoretograms.
Authors: Karinkanta H H , Nieminen E J
Published: January 2020

Immunofixation coupled with electrophoresis is winning favor as an easy, sensitive, and versatile method for detecting specific protein fractions. Here, we discuss problems which appear when antisera are applied onto the electrophoretic medium (Cellogel or cellulose acetate). Longitudinal slits can be made in the electrophoretic medium without worsening resolution. Such longitudinally slitted electrophoretic membranes can be used in electrophoresis and specific antisera applied simply by pipetting them onto the electrophoretograms. The method has been used successfully in typing immunoglobulins.
Performance of the Du Pont aca ammonia method.
Authors: Doumas B T , Hause L L , Sciacca R D , Jendrzejczak B , Foreback C C , Hoover J D , Spencer W W , Smock P L
Published: January 2020

We evaluated the performance of the Du Pont aca ammonia procedure with regard to (a) linearity, (b) precision, (c) interferences, and (d) effect of anticoagulants. Linearity extends to 2,000 mumol/L. The SD of the method was essentially constant (2 to 3 mumol/L) and independent of the NH3 concentration. Hemoglobin, bilirubin, and lipemia do not interfere. Either EDTA or heparin is suitable as anticoagulant. Recovery of NH3 added to plasma samples averaged 102% (range: 97--107%). We established normal values by measuring NH3 in 188 plasma samples from apparently healthy individuals. The 95% confidence range is from 10 to 35 mumol/L. The aca ammonia method compares very well with that of Kingsley and Tager but correlates less strongly with that of Reinhold and Chung. We describe a protein-based solution with stable NH3 concentration that is suitable as a control material.
Chemical inhibition used in a kinetic urease/glutamate dehydrogenase method for urea in serum.
Authors: Sampson E J , Baird M A
Published: January 2020

We describe a fixed-time-interval, kinetic inhibition method, with use of a competitive inhibitor (l) of the urease/glutamate dehydrogenase reaction to increase the "apparent" Michaelis constant by a factor of (1 + [l]lKl). This allows greater flexibility in selecting an appropriate sample dilution for kinetic determinations of urea in serum (i.e., [S]lKm ratio). Nine compounds were screened as potential inhibitors for this study. Adding 5 mmol of hydroxyurea per liter increases the "apparent" Michaelis constant for the coupled enzyme reaction by 10-fold. We used a sample dilution of 21-fold vs. dilutions of 141- to 350-fold for previously reported kinetic methods. Mean analytical recovery with this method was 100.2%. Reaction rate vs. urea concentration was linear, and complete recovery extended to 30 mmol of urea per liter. Of 22 potential interferents, only fluoride (250 mmol/L) and bilirubin (1 mmol/L, or 580 mg/L) caused greater than 5% interference. We discuss precision and effects of specimen dilution, and compare results for 100 specimens with those by a manual Berthelot-indophenol method, a manual diacetyl monoxime method, and a diacetyl monoxime method adapted to continuous-flow analysis.
Rate nephelometric measurement of rheumatoid factor in serum.
Authors: Finley P R , Hicks M J , Williams R J , Hinlicky J , Lichti D A
Published: January 2020

We describe the measurement of rheumatoid factor in human sera with a rate nephelometer. The National Reference Preparation for Rheumatoid Factors is used to calibrate the assay in International Units. We used Hyland Positive Control, Level I, as a secondary standard. The standard curve is exponential, but is linear when plotted on log-log graph paper. Aggregated immune globulin (IgG) is the antigen used to detect rheumatoid factor (IgM-class antibody to IgG). The rate reaction measures the rate of increase in light-scatter by the antigen-antibody complexes; the reaction takes place in 17 to 20 s. Precision, linearity, and accuracy are excellent. Results agree well with those for a commonly used latex precipitation test. The advantages of speed, quantification in International Units, and superior discrimination of concentration as compared to serological titration provide a more reliable test for use in the diagnosis and treatment of rheumatoid arthritis.
Re-evaluation of turbidimetry of proteins by use of aromatic sulfonic acids and chloroacetic acids.
Authors: Ebina S , Nagai Y
Published: January 2020

From studies on 11 different proteins (including native albumin and albumin with reduced disulfide-bridges) treated with sulfosalicylic, 2-naphthalenesulfonic, toluenesulfonic, dichloroacetic, or trichloroacetic acids, we elucidate the interactions determining the resulting turbidities and other factors affecting turbidities, and we discuss the clinical utility of such turbidimetry. At least three interactions are important in determining turbidity: reduction of positive charges on the protein, hydrogen bonding of the non-ionized chloroacetic acids with the protein, and hydrophobic interaction of the aromatic sulfonic acids with albumin. Turbidity varies appreciably with the species of acid and protein, concentrations of acid, temperature, and standing time after acid is added. We conclude that this technique should be restricted to confirming proteinuria.
An "antibody electrode," preliminary report on a new approach in enzyme immunoassay.
Authors: Boitieux J L , Desmet G , Thomas D
Published: January 2020

We report preliminary experiments on a new, sensitive, and reliable procedure for enzyme immunoassay of various antigens in biological fluids. The method, developed from the biological model "hepatitis B surface-antigen/antibodies," is less time consuming than most immunochemical techniques and eliminates many inconveniences arising from use of isotopes. We use a solid-phase "sandwich" procedure, the antibodies being immobilized on gelatin membranes, and determine antigen concentration with the help of an iodide-sensitive electrode modified by fixing the active membrane onto the crystal sensor. We have established the analytical criteria of the method and compared it with the solid-phase radioimmunoassay for the surface antigen in dilution series. One-tenth microgram of antigen per liter can be reproducibly detected with our method. Use of antibody electrode should easily be extended to assay of other antigens and haptens that usually are determined by radioimmunoassay.
An evaluation of ethylene glycol-based liquid specimens for use in quality control.
Authors: Pope W T , Caragher T E , Grannis G F
Published: January 2020

We investigated the analytical acceptability of ethylene glycol-based control sera by preparing sets of aqueous and ethylene glycol-based specimens that had added uric acid, calcium, creatinine, glucose, urea, sodium, and potassium. Ethylene glycol caused a significant systematic proportional bias in procedures involving dialysis, but had no discernible effect on methods not involving dialysis. The extent of bias was proportional to the concentration of ethylene glycol, was independent of protein concentration, and differed according to the type of dialysis system used. We conclude that ethylene glycol-based control materials can have useful applications in clinical chemistry, but caution must be exercised in their use with analytical systems that employ dialysis.
Multi-channel, probe colorimeter for use with the micr-ELISA test, which makes use of disposable flat-bottom microhemagglutination plates.
Authors: McMurray C H , Blanchflower W J
Published: January 2020

We describe a multiprobe colorimeter that permits the reading of enzyme-linked immunosorbent assays (ELISA) in situ, by use of microhemagglutination plates. The use of dipping probes eliminates interference from meniscus effects and air-bubble entrapment. Detailed validation tests have been done and potential sources of inaccuracy identified.
Method for determining thiocyanate in serum and urine.
Authors: Lundquist P , Mårtensson J , Sörbo B , Ohman S
Published: January 2020

We describe a method for rapid and specific measurement of thiocyanate in serum or urine. We separate thiocyanate from interfering compounds by adsorbing it on an anion-exchange resin that has special affinity for thiocyanate, then eluting with sodium perchlorate. The eluted thiocyanate is quantified by a modified König reaction, sodium hypochlorite being used as the chlorinating reagent. Analytical recovery of thiocyanate added to serum and urine was quantitative; the coefficient of variation was 2.3% for both within-day and between-day precision. Cyanide and certain antibiotics interfere, but may be eliminated by including additional washing steps in the usual procedure. The proposed procedure was compared with another method, based on the oxidation of thiocyanate to cyanide. Agreement was satisfactory, both for serum and urine.
Anaphylaxis in the mouse--a model for using two-dimensional electroimmunodiffusion to study pathological changes in multiple serum proteins simultaneously.
Authors: Quintero S , Crowle A J
Published: January 2020

Two-dimensional electroimmunodiffusion is rarely used in clinical chemistry, because its potentialities are not yet widely appreciated. Studying mouse anaphylaxis, we observed with this technique certain striking short- and long-term pathologic changes in several important serum antigens, which aptly demonstrate its uniqueness and potential clinical usefulness. We present some of these observations here, for illustration. We analyzed sera from CF-1 female mice that had been anaphylactically shocked with chicken conalbumin antigen and bled 45 min to 22 days afterward. In three separate experiments, with samples of serum being analyzed only once each, this technique easily and surely detected quantitative and (or) qualitative changes in several serum antigens. We discuss here the changes in 7S gamma1 immunoglobulin, C3 globulin, hemopexin, and alpha lipoprotein, and explain how some of these changes would have been difficult and others impossible to detect by currently routine tests. We urge the adoption of two-dimensional electroimmunodiffusion for frequent use in the clinical laboratory for diagnosis, prognosis, and monitoring pathological conditions.
Direct determination of mercury in blood by use of sodium borohydride reduction and atomic absorption spectrophotometry.
Authors: Sharma D C , Davis P S
Published: January 2020

A method is presented for the rapid determination of total mercury in blood. The reagent used is alkaline sodium borohydride, and no digestion of the sample is needed. The detection limit and sensitivity are 1.15 and 1.59 ng of Hg, respectively. The method gives reproducible results (the CV ranged from 5.3 to 6.7% for low and high mercury blood samples, respectively), comparable to those obtained by the digestion method. The mean analytical recoveries of added mercuric chloride and methylmercuric chloride were 106.65 and 99.02%, respectively. Other advantages of the method are freedom from contamination encountered with digestion methods and elimination of matrix effects.
Hyperamylasemia and ovarian carcinoma.
Authors: Sandiford J A , Chiknas S G
Published: January 2020

We present a case of advanced ovarian carcinoma associated with high concentrations of serum and urine amylase. Biochemical analysis to elucidate the nature and origin of the hyperamylasemia identified seven amylase isoenzymes, of which five appeared to be distinct from normal salivary and pancreatic amylase. We discuss the nature of amylase isoenzymes produced by malignant tumors.
Isoelectric focusing of serum proteins on modified thin cellulose-acetate membranes.
Authors: Ambler J , Walker G
Published: January 2020

We applied a method previously described for treating cellulsoe acetate gel strips with boron trifluoride in methanol, to make them suitable for isoelectric focusing of proteins, to dry cellulose acetate membranes. The results obtained are shown to compare very favorably. Both wide and narrow pH gradients may be used and coupled either to conventional protein staining or to specific identification by immunofixation. Focusing ordinarily takes 90--120 min, is technically easy, and is economical in the use of ampholytes.
IgD myeloma protein with "unreactive" light chain determinants.
Authors: Cejka J , Kithier K
Published: January 2020

Serum from a patient with multiple myeloma showed a monoclonal protein, classified by immunoelectrophoresis as IgD. Immunofixation electrophoresis and immunoelectrophoresis failed to demonstrate a precipitation reaction between the paraprotein and antisera to immunoglobulin light chains. The light chains of the monoclonal protein, immunologically inaccessible in the intact molecule, reacted with anti-lambda chain antisera only after reduction and alkylation of the paraprotein. Moreover, interpretation of the immunoelectrophoretic patterns was hampered by the presence in patient's serum of free lambda chains having about the same mobility as that of the paraprotein.
Solid-phase radioimmunoassay with protein-A-bearing Staphylococcus aureus cells used to assay a protein (ferritin) and a hapten (digoxin).
Authors: Gauldie J , Tang H K , Corsini A , Walker W H
Published: January 2020

We compared use of protein-A-containing Staphylococcus aureus bacteria with conventional ammonium sulfate precipitation and second-antibody methods of separating bound and free antigen in the radioimmunoassay of a hapten (digoxin) and protein (ferritin) in human sera. In each case, values obtained with the heat-killed, formalin-fixed bacteria correlated well with those found by established methods. No matrix effects were detected in either hapten or protein measurements. Because of the affinity of S. aureus for rabbit IgG, rabbit antisera could be used with a small number of bacteria to detect antigen in the presence of 50-fold excess human IgG. The availability of S. aureus and ease of handling make this reagent a rapid, economical alternative of general applicability in radioimmunoassay.
Extraction and determination of chloroform in rat blood and tissues by gas chromatography-electron-capture detection: distribution of chloroform in the animal body.
Authors: Vogt C R , Liao J C , Sun A Y
Published: January 2020

We have developed a simple, sensitive, and accurate method for the determination of chloroform in rat blood, brain, kidney, liver, and fat. The detection limit is 2.5 ng of chloroform per gram of tissue. Studies of in vivo distribution of chloroform in rat blood and target tissues after intragastric intubation of chloroform/water show that the amount of chloroform accumulated in the different tissues increases with increasing doses. Fat tissue contains the greatest amount of chloroform. The accumulation of chloroform in rat blood and target tissues seems to be maximum 1.5 h after administration, and the apparent chloroform concentration is almost at baseline value 8 h later.
Immunoglobulins in cerebrospinal fluid in various neurologic disorders.
Authors: Bouloukos A , Lekakis J , Michael J , Kalofoutis A
Published: January 2020

We determined the concentrations of immunoglobulins A, G, and M in cerebrospinal fluid of 16 patients suffering from multiple sclerosis, 13 with non-bacterial meningitis, 10 with stroke syndrome, and 13 with epilepsy. The differences in concentrations of immunoglobulins in these groups were remarkable in the patients with multiple sclerosis, meningitis, or stroke syndrome. We propose that the determination of the absolute immunoglobulin content in cerebrospinal fluid is of greater significance than the relative immunoglobulin concentration.
Continuous-flow analysis for glucose in serum, with use of hexokinase and glucose-6-phosphate dehydrogenase co-immobilized in tubular form.
Authors: León L P , Chu D K , Snyder L R , Horváth C
Published: January 2020

Hexokinase and glucose-6-phosphate dehydrogenase, co-immobilized at the inner wall of plastic tubing, were used with continuous-flow analyzers to measure serum glucose. The performance of the enzyme-containing coils meets the hydraulic requirements of the continuous-flow instruments, and the average carryover does not exceed 2.2%. A glucose concentration change of 1 g/L yields a 0.08 change in absorbance. There is no significant deviation from linearity for glucose concentrations up to 5 g/L. The method is similar to the Reference Method proposed by the Food and Drug Administration, and results by the two methods agree and correlate well (r = 0.999). The operational stability of the enzyme tubes is sufficient to allow the analysis of at least 10 000 serum samples. We investigated in detail the effect of reagent concentrations and temperature on the performance of the enzyme coils, and the results shed light on the nature of the heterogeneous dual-enzymic reaction.
Response of ion-selective sodium and potassium electrodes in the Beckman Astra 4 and Astra 8 analyzers.
Authors: Fievet P , Truchaud A , Hersant J , Glikmanas G
Published: January 2020

We tested the response of ion-selective sodium and potassium electrodes that are a part of the new multi-channel chemical analyzers, Astra 8 and Astra 4 (Beckman). For this we used plain and albumin-containing aqueous solutions of known Na+ and K+ concentrations, varied inversely so we could assess how the concentration of each electrolyte would affect the response of the other electrode. The stability of the electrodes with time was good and the electrodes are indeed selective for Na+ and K+.
Rapid preparation of nucleotides from acid-soluble pools by chromatography on silica, as exemplified with acid extracts of cultured cells.
Authors: Lothrop C D , Uziel M
Published: January 2020

A rapid technique (5-10 min) has been developed for fractionating nucleotides from base and nucleoside contaminants in acid extracts of cells, by adsorption to silica gels. Silica gels (1-mL bed volume) were washed with 5 mL of water then with 5 mL of acetonitrile/water (90/10 by vol). After applying 3-mL samples, adjusted to 900 mL/L acetonitrile content, we washed the gel with an additional 10 mL of the acetonitrile/water solvent. More than 95% of the amounts of bases and nucleosides prsent, except for cytidine (92%), did not adsorb to silica under these conditions. Nucleotides were then quantitatively eluted with 9 mL of water. The retention volumes for positive, negative, and neutral nucleic acid components have been determined, to investigate the discriminatory properties of nucleic acid components on silica. Compounds (bases, nucleosides) that are not ionized at pH 7 do not bind to silica. However, negative, positive, and zwitterionic compounds are tightly adsorbed to the silica gels. This procedure has been used to purify nucleotides from several normal and transformed cell lines.
Concentrations of magnesium, calcium, potassium, and sodium in human heart muscle after acute myocardial infarction.
Authors: Speich M , Bousquet B , Nicolas G
Published: January 2020

Atomic absorption spectrometry was used to measure magnesium, calcium, and sodium, and emission spectrometry to measure potassium, in myocardium (left and right ventricles) of 26 control subjects who died of acute trauma. Results were expressed in mumol/g of proteins. Mg/Ca and K/Na ratios were also determined. The same measurements were made in 24 patients who died from acute myocardial infarction. Samples were also taken from the necrotic area. Mg/Ca and K/Na ratios were significantly higher in the left ventricle of both populations, thus providing evidence of anatomical and physiological differences between the two ventricles. As a result of cytolysis and anoxia, the Mg/Ca ratio was very significantly inverted, and the K/Na ratio very significantly smaller, for samples from the necrotic area. In these clinical conditions arrhythmias could certainly be considered likely, and there is reason to believe that magnesium depletion may be a cause of arrhythmias.