bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative two-dimensional protein electrophoresis for studies of inborn errors of metabolism.
Authors: Merril C R , Goldman D
Published: January 2020

High-resolution electrophoretic methods and sensitive protein-detection techniques permit new approaches to understanding and diagnosis of the inborn errors of metabolism. These approaches encompass: the search for protein alterations that represent primary mutations effects; observation of alterations in protein patterns due to secondary effects, as might occur in major metabolic pathway abnormalities; and identification of protein polymorphisms that are genetically linked to an inborn metabolic disease. With the aid of computer analysis of the electrophoretograms, all three approaches are being developed. Protein density and position are evaluated with an interactive computer program that requires that gel polypeptides be indexed by the investigator. Proteins on the gels are made visible with an inexpensive, rapid silver stain, which can be used quantitatively. The Lesch-Nyhan syndrome, one of a few neuropsychiatric diseases for which the molecular defect is known, was chosen for study with these techniques. Four hundred proteins were analyzed for positional or quantitative variation. Eleven significant (2p less than 0.01) quantitative differences were found in autoradiograms from gels of phytohemagglutinin-stimulated lymphocytes. Specific patterns of polypeptide variation are now being sought in an expanded clinical study primarily focusing on Huntington's disease. Large studies are required to establish the specificity of observed alterations. As the number and variety of analyses increase, a correlative catalog of molecular variation and polymorphism will be generated.
Lymphocyte proteins in Huntington's disease: quantitative analysis by use of two-dimensional electrophoresis and computerized densitometry.
Authors: Goldman D , Merril C R , Polinsky R J , Ebert M H
Published: January 2020

We used quantitative two-dimensional electrophoresis to study lymphocyte proteins in Hungtington's disease. Three hundred and six polypeptides from 14C-labeled, phytohemagglutinin-stimulated lymphocytes were measured for variation in relative spot density and 186 for variation in spot position by use of a computer program requiring operator interaction. Each polypeptide was measured in a total of 30 electrophoretograms from 28 individuals, including 13 with Huntington's disease, 2 at risk for it, and 13 controls. The study included two sets of identical twins and, as neurological controls, individuals with neurofibromatosis, Alzheimer's disease, or Shy-Drager syndrome. Seven protein polymorphisms were identified among the 186 most dense polypeptides of each gel, corresponding to a minimum average heterozygosity of 1.4%. Stringent criteria were used to define polymorphic proteins, including observation of at least one individual with each of two homozygous phenotypes and one with the heterozygous phenotype, demonstration of the expected gene dosage relationship by quantitative densitometry, consistency with genetic relationships, and reproducibility. One polymorphic protein showed three electrophoretically variant alleles. Our identification of seven polymorphisms among the 186 proteins measured on a single electrophoretogram illustrates the potential of this technique for performing linkage analysis in diseases of genetic origin. However, we observed no quantitative or positional protein variations that were characteristic of (i.e. specific for) Huntington's disease.
Two-dimensional electrophoresis in basic and clinical research, as exemplified by studies on the exocrine pancreas.
Authors: Scheele G A
Published: January 2020

I summarize our experience in the development and use of two-dimensional isoelectric focusing/SDS gel electrophoresis for the study of soluble proteins, in particular those of the exocrine pancreas. The importance of adding protease inhibitors to the first-dimension isoelectric focusing gel to prevent the autoactivation of potential pancreatic proteases and subsequent proteolysis is discussed. Recently developed methods for both the indirect and direct identification of protein spots by biological activity are summarized. I give examples of the importance of this technique in the study of basic mechanisms involved in the synthesis, segregation, and secretion of exportable proteins in the exocrine pancreas, as well as in the search for biochemical markers of pancreatic disease.
Two-dimensional analysis of human lymphocyte proteins II. Regulation of lymphocyte proteins by a molecule present in normal human urine.
Authors: Willard K E
Published: January 2020

Lymphocytes cultured with concentrated human urine develop many alterations (Clin. Chem. 27:1327, 1981). New proteins appear ("Urocon" proteins), while others disappear ("Urocof" proteins) from the two-dimensional gel pattern. This paper characterizes a human urinary leukocyte effector protein ("HULEP") that is responsible for the appearance of the Urocon:11-15 proteins in human lymphocytes. The effector molecule, HULEP-1, is a protein with a relative molecular mass in the range of 29000 to 43000. Urocon:11-15 production, cytoskeletal changes in the lymphocyte, and the presence of added HULEP-1 in the cultures are all directly related. This molecule appears to alter the structure of the lymphocyte by degrading actin and possible other lymphocyte cytoskeletal proteins. Peptide maps of Urocon:11-15 and beta and gamma actin provide evidence that Urocon:11-15 proteins are closely related to nonmuscle actin, and may be degradation products of this major cytoskeletal protein.
Determination of lipids, including medium-chain fatty acids, in human feces.
Authors: Esteves W , Soares L A , Hartman L , Pereira A S
Published: January 2020

In this method for determining fecal lipids, feces are saponified with ethanolic potassium hydroxide, the ethanol is evaporated, and the acidified residue extracted with hexane. An aliquot of the hexane solution is then titrated with ethanolic sodium hydroxide, the solvents are evaporated, and the residue (soaps and unsaponifiable matter) is weighed. The unsaponifiable matter is extracted with acetone and determined gravimetrically. The method thus permits the determination of milli-equivalents of fatty acids, total lipids, and fatty acids in one sample and is applicable to feces containing medium-chain fatty acids and their triglycerides.
Simple, direct measurement of lipoprotein X in serum.
Authors: Hashiguchi Y , Kawaguchi M , Nakanishi M , Matsumori Y , Kaneda Y , Nakai C , Mimura K , Matsuoka A
Published: January 2020

This relatively simple method for quantitative estimation of lipoprotein X is based on assay of phospholipids in precipitates of lipoprotein X. We first remove other lipoproteins by precipitation with phosphotungstic acid solution. Lipoprotein X is then precipitated from the supernatant fluid with an alkaline solution of magnesium ion, and phospholipid is determined in this precipitate. Results are linearly related to concentration of lipoprotein X. The CV is less than 3%. Results correlated well with those for agar gel electrophoresis.
Macro creatine kinases: results of isoenzyme electrophoresis and differentiation of the immunoglobulin-bound type by radioassay.
Authors: Bohner J , Stein W , Steinhart R , Würzburg U , Eggstein M
Published: January 2020

In 2.9% of sera from 1253 unselected patients we detected two different types of macromolecular creatine kinases (CK; EC 2.7.3.2). One macro type was represented by immunoglobulin-linked CK: in sera containing macro CK-BB isoenzyme, 125I-labeled CK-BB was bound with high affinity to the immunoglobulin fraction. Furthermore, during electrophoresis, macro CK-BB mostly migrated between CK-MB and CK-MM, and was fixed to Protein A from Staphylococcus aureus. We therefore propose radioelectrophoresis as a specific, highly sensitive, and simple method for detecting this type of macro CK. This form occurs predominantly in elderly women, is not correlated to any specific disease, and persists in blood over a long period of time. In contrast, a second type (macro-CK type 2) never bound radiolabeled CK isoenzymes, and was not adsorbed to Protein A. Electrophoretic migration of this macro-CK type 2 was generally cathodic to CK-MM. We observed this type in severely ill patients, frequently those suffering from malignant tumors. Clinical observations and biochemical data suggest that macro-CK type 2 is of mitochondrial origin.
Gas-chromatographic determination of verapamil and norverapamil, with a nitrogen-selective detector.
Authors: Vasiliades J , Wilkerson K , Ellul D , Anticoli M , Rocchini A P
Published: January 2020

We present a procedure for the determination of verapamil and its metabolite, norverapamil, in serum. The drugs are extracted under basic conditions into n-heptane/2-butanol (96/4 by vol) and then extracted again into 1 mol/L H2SO4. The acidic solution is made basic with sodium hydroxide, re-extracted with diethyl ether, and the extract evaporated. The residue is redissolved in ethanol and analyzed by gas chromatography with a nitrogen-selective detector. By use of two internal standards, prazepam and D-517 (a verapamil analog), concentration and instrument response are linearly related from 50 micrograms/L to 5 mg/L. Within-run precision (CV) was 3 and 5% for both verapamil and norverapamil at concentrations of 100 and 250 micrograms/L; between-run precision was 11 and 9% at those respective concentrations. Interference studies indicate that most commonly prescribed basic drugs will not interfere with this procedure.
Simultaneous fluorometry of urinary dopamine, norepinephrine, and epinephrine compared with liquid chromatography with electrochemical detection.
Authors: Oka K , Sekiya M , Osada H , Fujita K , Kato T , Nagatsu T
Published: January 2020

A simple fluorometric method is described for simultaneous assay of urinary norepinephrine, epinephrine, and dopamine. The catecholamines are separated by boric acid gel chromatography and subsequently determined by use of a modified trihydroxyindole reaction. By the separation with boric acid gel, dopa can be eliminated, which interferes with dopamine measurement; consequently, the reaction is specific for catecholamines. This simple, rapid method was applied to urine specimens from normal adults. Values for urinary dopamine, norepinephrine, and epinephrine agreed with those obtained by "high-performance" liquid chromatography with electrochemical detection.
Danazol and its principal metabolites interfere with binding of testosterone, cortisol, and thyroxin by plasma proteins.
Authors: Haning R V , Carlson I H , Cortes J , Nolten W E , Meier S
Published: January 2020

Danazol and its three principal metabolites (2-hydroxy-methylethisterone, 2-hydroxymethyl-1,2-dehydroethisterone, and ethisterone) competitively displace cortisol and testosterone from plasma proteins. This effect is in addition to the reported inhibition of the production of testosterone-binding globulin and thyroxin-binding globulin. We saw no competitive inhibition of thyroxin binding. Concentrations of total testosterone, total cortisol, and total thyroxin were low, whereas percentages of free testosterone, free cortisol, and free thyroxin were abnormally high in women being treated with danazol. Values for testosterone, cortisol, and thyroxin in danazol-treated patients should therefore be appropriately corrected before interpretation. Protein-binding assays for testosterone or cortisol that involve testosterone- or cortisol-binding globulin may be invalid in danazol-treated subjects because of the competitive binding of danazol and its metabolites to these proteins.
Versatile semiautomated sample processor and gamma counter to increase radioimmunoassay efficiency.
Authors: Vihko R , Bolton N , Hammond G L , Kankkunen C , Koskinen L , Martio A , Soini E , Vaintola R
Published: January 2020

We describe a semiautomated batch-assay system designed to increase the speed and ease of performing radioimmunoassays while maintaining good accuracy and precision. The main components are a programmable pipetting unit (sample processor) and a gamma counter capable of simultaneously counting radioactivity in 12 samples. The processor uses prearranged sets of as many as 24 disposable pipette tips, which eliminates carryover between samples, and features a horizontal shaker and magnetic stirrer; 50 different assay protocols can be selected. Batches of tubes are easily transferred from the sample processor to the incubator and centrifuge. The gamma counter is controlled by a microprocessor, which is also used for data processing. Because the system is based on discrete rather than continuous-flow analysis and because of the ease with which consecutive samples can be processed, more than one operation and several different assays can be accommodated simultaneously, which saves time considerably.
Kinetic assay of beta-hydroxybutyrate in plasma with a COBAS-BIO centrifugal analyzer.
Authors: Moore J J , Marcus M , Sax S M
Published: January 2020

An enzymic rate method for measuring beta-hydroxybutyrate in plasma by use of beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) was adapted to the Roche COBAS-BIO centrifugal analyzer. Optimum reagent composition and reaction conditions were determined, to provide increased sensitivity and accuracy. Use of magnesium ions increased sensitivity. Comparison of results with those of a manual enzymic endpoint method involving perchloric acid protein-free filtrates of whole blood showed excellent correlation (r = 0.996). The method has good within-run and day-to-day precision. Linearity of the standard curve extends to 5000 mumol of beta-hydroxybutyrate per liter. The assay can be completed in less than 5 min and requires 15 microL of plasma and minimal reagent volumes.
Advantages of Use of Ascorbic Acid and Sodium Lauryl Sulfate in the Microdetermination of Serum the Inorganic Phosphorus
Authors: Bingley J B , de Postiglione M D G T
Published: January 2020

Vol. 27

p 639: In the second paragraph of the middle column, the third sentence (et seq.) should read as follows: In the method of Chen et al. (12), modified to include SLS 1,040 mmol/L, 25 µL of serum was added to 25 µL of distilled water. To this was added 200 µL of a solution of SLS containing 1040 mmol/L, followed, after mixing, by 2.50 mL of a reagent consisting of 3.1 g of ammonium molybdate per liter of 375 mmol/L sulfuric acid. To avoid any instability in the reducing capacity of ascorbic acid if mixed in advance with molybdate and sulfuric acid (14), we added it separately—0.200 mL of a 150 g/L solution in glass-distilled water. After incubation in a warm bath at 37 °C for 140 min (12), the replicated stoppered samples were cooled and their absorbances (820 nm, 1-cm cells) read immediately and 3 and 18 hlater. In those cases where SLS was not used—i.e., in the unmodified method (12)—50 µL of a prepared trichloroacetic acid extract, equivalent to 25 µL of serum, was analyzed in the same manner; 200 µL of distilled water then replaced the SLS.

p 1401: In footnote 1, the compound should read N1-(α,α-dimethyl - β - hydroxyethyl) - 2- methyl - 1,2- propanediamine.

p 1749: In Table 3, column 4, row 2: "0-20" should read "0-2.0."

p 1948: In ref. 12, "Sud-" should read "Sul-" (publication is in Portugese, not Spanish).

p 1952: The names of two of the authors are misspelled: Cassani and Vanzetti. The correct spellings in the address are "Granda" and "Niguarda."

Vol. 28

p 156: In Table 1, last column, delete "x" from the equations "y = 0.75x" and "y = 1.71x."

p 401: In the address in the middle column, "State" should read "Safe."

p 406: Third column, under "Natelson Award," Normal should read Norman.

p 557: Just before the last sentence in the second paragraph in the middle column, insert the following sentence: "The exact degree of the enhancement depends highly on the amount of catalase impurity in the glucose oxidase preparation."
A simple method of eluting proteins from two-dimensional gels.
Authors: Guevara J , Chiocca E A , Clayton F C , von Eschenbach A C , Edwards J J
Published: January 2020

Apo A-I lipoprotein was extracted from the appropriate pieces of two-dimensional gel patterns, by electrophoresis onto hydroxylapatite overlaying a slab of polyacrylamide sodium dodecyl sulfate gel. The extracted protein was then eluted from the hydroxylapatite by washing with increasing concentrations of phosphate buffer. The recovered protein was electrophoretically homogeneous. We believe that this method can be generally useful in the purification, for study, of proteins identified on two-dimensional gels as putative markers of disease.
Enhancement techniques for detecting trace and fluid-specific components in two-dimensional electrophoresis patterns.
Authors: Dermer G B , Silverman L M , Chapman J F
Published: January 2020

Albumin and other serum-derived proteins were removed from several types of body fluids by affinity chromatography, to facilitate detection of trace or non-serum-derived proteins in two-dimensional electrophoresis patterns. Albumin was removed by the dye Cibacron Blue F3G-A coupled to Sepharose. Two-dimensional patterns of albumin-depleted serum lack the large albumin spot, and several families of spots become visible that ordinarily are partly or totally hidden by it. However, other proteins also bind to Cibacron Blue. Most serum proteins, including albumin, were effectively removed by anti-human serum antibodies coupled to Sepharose. Two-dimensional patterns of serum-depleted cerebrospinal fluid exhibit five clusters of probable nervous-system protein families not detected in serum. One additional family, probably antigenically related to transferrin, was removed by the affinity step. Two-dimensional patterns of serum-depleted prostatic fluid exhibit five major non-serum families, two of which may be creatine kinase B subunits and prostatic acid phosphatase. Two-dimensional patterns of serum-depleted malignant effusions exhibit one or more of three proteins that possibly are tumor products. Pattern matching suggests the presence of one non-serum-derived protein family common to cerebrospinal fluid, prostatic fluid, and malignant effusions. Prostatic fluid and malignant effusions have in common as many as three non-serum families of proteins.
Two-dimensional gel electrophoresis of human brain proteins. II. Specific proteins and brain subfractions.
Authors: Comings D E , Carraway N G , Pekkula-Flagan A
Published: January 2020

Brain proteins extractable with distilled water or 9 mol/L urea were subjected to two-dimensional gel electrophoresis. They are examined in relation to the identification of actin, tubulin, neurofilament proteins, glial fibrillary acidic protein; proteins of human embryonic neocortex, synaptosomes, myelin, and plasma; and rat neocortex, rat glial, and mouse neuroblastoma cell lines.
Two-dimensional gel electrophoresis of human brain proteins. V. Non-equilibrium gel electrophoresis, with detection of a myelin basic protein mutation--MBL-Duarte.
Authors: Comings D E , Pekkula-Flagan A
Published: January 2020

To examine the basic human brain proteins, we subjected 9 mmol/L urea extracts to non-equilibrium gel electrophoresis. The pattern observed differs distinctly from that with equilibrium gel electrophoresis. With this technique, the myelin proteins (myelin basic protein, proteolipids, and basic Wolfgram proteins) and many other unindentified major basic proteins can be demonstrated. The myelin basic proteins occur as two major polypeptides of different charge and slightly different molecular mass, indicating the action of at least two genes. The proteolipid proteins occur as a long series of charge isomers, suggesting multiple genes or extensive post-transcriptional modification. In one patient with schizophrenia, a charge-change mutation of the larger myelin basic protein (MBL) was observed and is termed "MBL-Duarte."
A normalized protein map of human plasma, and characterization of the normalized spots by microscale amino acid analysis.
Authors: Manabe T , Kojima K , Jitzukawa S , Hoshino T , Okuyama T
Published: January 2020

A "normalized map" of human plasma proteins, which shows the standard distribution pattern of the proteins separated two-dimensionally in the absence of denaturing agents, is described. The pI and molecular mass of the protein represented by each spot in the normalized map are shown, and 84 of 128 spots are identified or tentatively identified as known plasma proteins. Of the several methods we used for the identification, the method of micro-scale amino acid analysis of the proteins extracted from the fixed, stained two-dimensional gels is described in detail. Patterns for human cerebrospinal fluid and urinary proteins are compared with the normalized map, the positions of proteins characteristic of each body fluid sample being shown.
Human proteins sensitive to neoplastic transformation in cultured epithelial and fibroblast cells.
Authors: Bravo R , Celis J E
Published: January 2020

We resolved the [35S]methionine polypeptides synthesized by normal [lung fibroblasts (WI-38), amnion cells] and transformed [SV40 transformed WI-38, AMA (spontaneously transformed amnion cells)] cultured human cells, using two-dimensional gel electrophoresis under conditions in which about 1300 polypeptides could be reproducibly separated. These studies demonstrated important changes in the relative proportions of several polypeptides that are present both in normal and transformed cells. Of a total of 400 common polypeptides that we quantitated for each cell type, 53 (22 basic and 31 acidic) varied by 40% of more in both cell pairs. Among these, we have identified vimentin (IEF 26), cyclin (IEF 49), and a tropomyosin-related polypeptide (IEF 52). No new major polypeptide was detected in the transformed cells, at least at the level of resolution currently achieved by this technique. Similar but qualitative studies of [32P]orthophosphate-labeled proteins revealed that, of 250 analyzed, only seven common phosphoproteins, including phosphovimentin (IEF 26e), varied consistently in both cell pairs. These results strengthen our previous conclusion that transformation results in changes in the relative proportions of polypeptides synthesized in normal and transformed cells rather than in the appearance of new polypeptides in transformed cells.
Two-dimensional gel electrophoresis method for investigation of human plasma proteins: detection of subtle changes during filtration leukapheresis.
Authors: Lonberg-Holm K , Bagley E A , Nusbacher J , Heal J M
Published: January 2020

Special problems are associated with analysis of human plasma proteins by standard "high-resolution" two-dimensional gel electrophoresis methods in which isoelectric focusing is followed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels (SDS-PAGE). Individual plasma proteins are often separated into overlapping groups of multiple spots, and identification of individual spots is further confounded by genetic variation. Analytical recovery of components of high molecular mass is also low or variable. These problems may be reduced or overcome by use of a "low-resolution" method consisting of electrophoresis of native proteins at pH 8.6 in an agarose gel followed by SDS-PAGE without added reducing agent. About 60 proteins can be resolved, most as single spots. About 25 of these proteins have been "mapped," and tentatively identified. We have examined 119 plasma samples taken from six donors who were undergoing filtration leukapheresis and 10 donors who were undergoing centrifugation leukapheresis or plateletpheresis. In all cases, passage of blood through a nylon filter induced a significant increase in a doublet of spots tentatively identified as complement component C3c. This was detected in the effluent from the filter throughout the first 30 min of filtration, and to a lesser extent in the venous blood. These spots were not induced by the centrifugation procedures. One filtration donor also showed increased acute-phase proteins 24 h after the procedure.
On the use and computation of likelihood ratios in clinical chemistry.
Authors: Albert A
Published: January 2020

The clinical relevance of likelihood ratios (L-values) for revising the physician's diagnostic probabilities has been recognized. However, the calculation of L-values, particularly in the case of quantitative or mixed quantitative-binary test results, raises problems that have not yet been addressed. Based on a very general assumption that yields a simple functional form for the likelihood ratio, a method is developed that allows such calculations regardless of the nature and the number of clinical laboratory tests to be interpreted simultaneously. Hence the notion of predictive value (posterior probability) is extended from binary or dichotomized tests to quantitative tests, and from univariate to multivariate clinical laboratory results. The simplicity and flexibility of this approach eliminates difficulties in computation arising from the addition of new data to an existing data base. It is hoped that this method will now allow L-values to be reported along with the original test results in daily laboratory practice.
Measurement of plasma estradiol-17 beta by solid-phase chemiluminescence immunoassay.
Authors: Kim J B , Barnard G J , Collins W P , Kohen F , Lindner H R , Eshhar Z
Published: January 2020

We describe a simple, solid-phase chemiluminescence immunoassay for the measurement of estradiol-17 beta in extracts of peripheral venous plasma. The method has similar sensitivity, specificity, precision, and accuracy to a conventional radioimmunoassay with a tritiated antigen. An immunoglobulin G fraction od monoclonal antibodies to estradiol-6-carboyxmethyl oxime-bovine serum albumin is passively adsorbed onto the walls of polypropylene tubes. The labeled antigen is estradiol-6-carboxymethyl oxime-aminobutylethyl isoluminol. After the binding reaction (1 h at 22 degrees C), the solution is removed by aspiration (400 microliters). Sodium hydroxide (5 mol/L, 300 microliters) is added and the mixture incubated for 30 min at 37 degrees C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10 s. The light yield is inversely proportional to the concentration of estradiol in the standard or sample.
A colorimetric assay for releasable plasminogen activator.
Authors: Campbell E E , Shifman M A , Lewis J G , Pasqua J J , Pizzo S V
Published: January 2020

We describe an equilibrium assay for measuring release of plasminogen activator form blood-vessel walls and report data from 125 individuals free of overt thromboembolic disease. Excess human plasminogen is added to the euglobulin fraction of plasma obtained before and after venous occlusion at mean systolic pressure. To measure plasmin generation in these samples, we used the chromogenic plasmin substrate D-Val-Leu-Lys-p-nitroanilide, which liberates p-nitroaniline upon cleavage. Releasable plasminogen activator in 24 subjects was determined by this colorimetric assay and by the radiocasein assay previously reported by this laboratory (Am. J. Clin. Pathol. 76,403-409, 1981), and the results were compared. The correlation coefficient was 0.97. The colorimetric assay offers several advantages over the radiocasein assay: shorter incubation (6 vs 16 h) and no preparation or quantification of a radioactive substrate and its cleavage products.
Application of response surface methodology to the assay of gamma-glutamyltransferase.
Authors: London J W , Shaw L M , Theodorsen L , Stromme J H
Published: January 2020

Response surface methodology (RSM) offers an empirical approach to the study of clinical enzyme assays. Variables such as pH, which are difficult to characterize by using theoretical enzyme kinetics, are easily included in RSM formulations. In this investigation, we studied with RSM the change in the measured activity of gamma-glutamy-transferase (EC 2.3.2.2) as a function of changes in concentrations of donor (gamma-glutamyl-3-carboxy-4-nitroanilide) acceptor (glycylglycine), and pH. The study defined large ranges for these variables over which maximum enzyme activity is obtained: donor 6.6 to 10.2 mmol/L, acceptor 129 to 250 mmol/L, and pH 7.8 to 8.5. The RSM regression polynomial was as accurate as a previously determined enzyme kinetic equation for predicting the transferase activity from given reagent substrate concentrations. Although not yielding a mechanistic understanding of an enzyme assay, RSM studies do produce an operational understanding of how an assay functions.
Creatine kinase isoenzymes in neonate plasma by cellulose acetate electrophoresis: albumin and adenylate kinase artifacts.
Authors: Massey T H , Barta J S
Published: January 2020

Patterns of creatine kinase (CK, EC 2.7.3.2) isoenzymes were studied in apparently healthy one- to 10-day-old neonates, by use of a sensitive fluorescent staining method with Sclavo CK-F/6001 reagent. Mean activities of CK3 (MM, 105 U/L), CK2 (MB, 6.8 U/L), CK1 (BB, 11 U/L), adenylate kinase (EC 2.7.4.3) anodal to CK3, and a fluorescent albumin artifact were found. Pooled plasma from neonates is recommended as a control because it defines the albumin artifact and approximates the activity of CK2 that must be observed after proper staining before a diagnosis of myocardial infarction can be made.
Theoretical and mathematical conditions for use of linearization methods for saturation-type radioassay data.
Authors: Shaw W
Published: January 2020

The algebraic equivalency of nine different methods for the linearization of saturation-type radioassay data was previously demonstrated mathematically to hold under the assumption that the antibody is saturated with ligand. Although compete saturation with antibody never occurs, a good approximation to a saturation condition occurs when the product of the labeled ligand and the affinity constant is 100 or more. The mathematical basis for determining when methods for the linearization of saturation-type radioassay data are valid is derived from a development of the first-order mass action law applied to ligand-antibody reactions.
Multiple myeloma protein with three light chains.
Authors: Sun T , Erichson R B , Lien Y Y , Wu C H
Published: January 2020

A case of immunoglobulin G (kappa) myeloma showed, in addition to the monoclonal IgG(kappa) arc, two kappa chains in the serum. The urine specimen contained 7.75 g of kappa chains per liter. The electrophoretically fast-moving kappa chain in serum was shown by immunoelectrophoresis and Ouchterlony immunodiffusion to be a complex of kappa chains and alpha 1-antitrypsin. This complex, which was detected only transiently in the patient's blood, was composed of a monomeric kappa chain bound to the antitrypsin by a disulfide bond. The predisposing factor for the formation of this complex is unclear, but patients showing this complex usually have kappa type myeloma protein and excrete kappa chain in urine at more than 1 g/L. The relationship between chemotherapy and formation of the kappa chain-antitrypsin complex needs further investigation.
Analytical artifacts in radioimmunoassay of L-thyroxin in human milk.
Authors: Mallol J , Obregón M J , Morreale de Escobar G
Published: January 2020

Previous results are contradictory regarding the concentration of thyroxin in human milk. Using a sensitive radioimmunoassay, we have found a lack of parallelism between the standard curve for thyroxin and the curve for serial dilutions of whole human milk, skimmed milk, or ethanol extracts of milk. Nonspecific binding also indicated the presence of analytical artifacts. Thus we have separated thyroxin from other milk components by means of a strongly basic Bio-Rad anion-exchange resin with quaternary ammonium exchange groups attached to a styrene divinyl benzene copolymer lattice, radioimmunoassaying the fractions eluted with an equivolume mixture of acetic acid and water. Parallelism with the standard curve was good, and results were the same whether or not the resin eluate was further purified by paper chromatography. The range of thyroxin concentration in 21 samples of human milk was 0.29-2.00 micrograms/L (mean 0.71, SD 0.40, microgram/L). Such concentrations are unlikely to afford protection to the developing brain of a breast-fed athyreotic baby, as previously claimed.
A micro-assay for estrogen receptor in breast tumor with use of 125I-labeled estradiol.
Authors: Van N T , Fritsche H A , Trujillo J M
Published: January 2020

The concentration of estrogen receptor protein in breast-tumor tissue is generally expressed in units of femtomoles of estradiol bound by the receptor per milligram of cytosol protein. The sensitivity of the estrogen receptor radioligand assay is therefore related to the specific activity of the steroid label used for the binding assay, the amount of the receptor protein in the volume of cytosol used, and the protein concentration in the cytosol. In this paper, we discuss factors affecting the sensitivity of the estrogen receptor assay and present various approaches for optimizing the assay. We also describe a procedure that involves a radioiodinated estradiol of high specific activity, a micro-technique for preparing tumor cytosol, and a micro-assay procedure with which the estrogen receptor protein can be measured in as little as 50 mg (wet weight) of tissue.
Comparison of calculated and measured free thyroid hormones in serum in health and in abnormal states.
Authors: Fresco G , Curti G , Biggi A , Fontana B
Published: January 2020

To evaluate a theoretical model for calculating free thyroid hormones, based on the law of mass action, we compared values for both calculated and measured free thyroxin and free triiodothyronine in a group of normal subjects. To determine whether the concentrations of circulating free hormones were also predictable from equilibrium considerations in abnormal states, we compared calculated and measured free thyroid hormone values in an additional population including pregnant women and hyperthyroid, hypothyroid, and "sick euthyroid" patients. Predictions based on the model were accurate in all these states except pregnancy, where there was some discrepancy between calculated and measured values for both hormones. In pregnant women with large abnormalities in thyroid-hormone-binding proteins, euthyroidism was accompanied by significantly lower free hormone concentrations, leading us to conclude that, in pregnancy, equilibrium may be reached at concentrations lower than those in other healthy subjects. Values for calculated and measured free thyroid hormones in "sick euthyroids" showed no discrepancy; however, we cannot exclude the possibility that non-dialyzable compounds are present that interfere with the hormone-protein binding.
Results by fluorescent immunoassay for phenytoin compared with those by enzyme immunoassay, liquid chromatography, and discrete analysis (Dupont aca).
Authors: Gonzalez G , Cid-Amador A , Steele B , Castro A
Published: January 2020

We describe the Ames Fluorescent Enzyme Immunoassay procedure for the detection of phenytoin in serum, and compare the results with it with those by enzyme immunoassay(EMIT), "high-pressure" liquid chromatography, and the Dupont aca. Sera from 48 patients who were receiving phenytoin were promptly analyzed in duplicate by EMIT, frozen, then assayed within four weeks by the other three methods. Correlation was good between results by the fluorescent immunoassay and the other methods. The fluorescent immunoassay procedure is sensitive and requires a small sample volume (100 microliters). Sample reading time can be shortened from 30 to 15 s as the technician becomes more proficient, with no sacrifice of accuracy. The procedure is fast, easy to perform, specific, sensitive, and inexpensive; involves no radioactivity; and requires no temperature control. It is the current method of choice for detection of phenytoin in serum, in terms of precision and accuracy.
A simple, fully enzymic bioluminescent assay for triglycerides in serum.
Authors: Björkhem I , Sandelin K , Thore A
Published: January 2020

In this fully enzymic bioluminescent assay, triglycerides are cleaved by lipase (EC 3.1.1.3) and carboxylesterase (EC 3.1.1.1), and the glycerol obtained is phosphorylated with ATP and glycerol kinase (EC 2.7.1.30). The ATP consumed in the latter reaction is determined by the firefly luciferin-luciferase reaction, and corresponds to the concentration of triglycerides. All the enzymic reactions and the bioluminescent reading can be performed in the same test tube. The precision (CV) of the assay varied between 1 and 7% at different concentrations with a fully enzymic method based on spectrophotometry (r = 0.98).
Liquid-chromatographic determination of pentobarbital in plasma with use of a resin column and an alkaline mobile phase.
Authors: Gupta R N , Smith P T , Eng F
Published: January 2020

We describe a liquid-chromatographic method involving a new, nonsilica column (XAD-2, Hamilton Co.) for pentobarbital in plasma. Plasma is extracted with chloroform after addition of the internal standard, 5-ethyl-5-p-tolyl-barbituric acid. Acidic drugs are back-extracted into alkali, then chromatographed on the resin-base reversed-phase column. The use of alkaline mobile phase allows enhanced sensitivity and detection of barbiturates at 240 nm. The within-run CV for 10 samples was 1.9%, the between-run CV 1.8%. Ten commonly used barbiturates are separated isocratically in less than 15 min. Other commonly prescribed acidic drugs do not interfere with determination of pentobarbital.
Improved direct determination of alpha- and gamma-tocopherols in plasma and platelets by liquid chromatography, with fluorescence detection.
Authors: Lehmann J , Martin H L
Published: January 2020

Tocopherols extracted from plasma with methanol or from platelets with chloroform/methanol were injected in methanol on a reversed-phase (C18) "high-performance" liquid-chromatographic column and eluted with water/methanol (2/98, by vol) at a flow rate of 1.4 mL/min. A "high-performance" spectrophotofluorometer was used for detection. Analytical recoveries ranged from 89 to 106%. The response was linear to at least 0.3 micrograms of either tocopherol (alpha- or gamma-) applied to the column, and the limit of detection was 0.1 ng. The method was used to measure tocopherols in plasma and platelets from human subjects, and some values are presented.
Enzyme immunoassay with flow-amperometric detection of NADH.
Authors: Eggers H M , Halsall H B , Heineman W R
Published: January 2020

Using phenytoin as a model analyte, we demonstrate that an enzyme-coupled immunoassay based on flow-injection analysis and amperometric detection of NADH is both feasible and practical. Good agreement with a routine clinical laboratory procedure for phenytoin was obtained for patients' serum samples. The electrode must be protected to prevent fouling by proteins in the analytical sample. The optimum detection range for NADH was at 0.01 of the concentrations of NADH generated during the several minutes required for each analysis. This suggests that the electrochemical technique should be extendable to the determination of species at concentrations in the microgram per liter range.
Radial partition immunoassay.
Authors: Giegel J L , Brotherton M M , Cronin P , D'Aquino M , Evans S , Heller Z H , Knight W S , Krishnan K , Sheiman M
Published: January 2020

In radial partition immunoassay, radial chromatography is used for performing an immunoassay. We describe the application of this technology to the measurement of digoxin in serum by enzyme immunoassay, with the entire testing procedure carried out on glass-fiber filter paper. A sample is applied to a small central area of the filter paper, where it reacts with the antibody to digoxin immobilized there. Subsequently, enzyme-labeled digoxin is applied to react with remaining antibody sites. After incubation, substrate for the enzyme is applied to the center of the reaction area and washes out any unbound label to the periphery of the paper. This step also initiates the enzyme reaction, which is quantified by front-surface fluorescence. A microprocessor-controlled automated instrument has been developed to process the filter paper matrix through the above sequence, and calculate the final result. Total testing time for digoxin is less than 7 min.
Development of the Biostator Glucose clamping algorithm.
Authors: Clemens A H , Hough D L , D'Orazio P A
Published: January 2020

The "glucose clamping" technique has been proposed as a method for the early detection of a beginning derangement of glucose homeostasis and thus for the possible prevention of maturity-onset diabetes. This technique interrupts the physiological glucose-insulin relationship by placing a patient's blood glucose concentration under an investigator's control, for quantification of the pancreatic beta-cell response during hyperglycemic clamps and of sensitivity of body tissue to exogenous insulin during normoglycemic clamps. We report the development of a glucose clamping algorithm for use with the Biostator glucose-controlled insulin-infusion system (Horm. Metab. Res., Suppl. 8: 23-33, 1977). This algorithm adds simplicity and precision to the glucose clamping procedure and reduces operator effort to a minimum. We describe the early development of the algorithm with a model system and report evaluations made during animal studies and preliminary investigations with human subjects.
Rapid enumeration of T lymphocytes by a flow-cytometric immunofluorescence method.
Authors: Ip S H , Rittershaus C W , Healey K W , Struzziero C C , Hoffman R A , Hansen P W
Published: January 2020

Combined with current advances in microprocessor-controlled flow cytometers, monoclonal antibodies provide a rapid means of phenotyping individual cell surface markers for a large number of clinical samples accurately and reproducibly, which may provide useful information in diagnosing disease and monitoring patients. We have developed a one-step flow-cytometric immunofluorescence procedure for enumerating E-rosette lymphocytes from whole blood by using the monoclonal antibody OKT11. This antibody recognizes the sheep erythrocyte receptor on the lymphocyte surface and can block sheep E-rosette formation. The flow cytometer we use, an Ortho Spectrum III, distinguishes lymphocytes from other leukocytes by measuring the narrow forward and right-angle light-scattering properties of the cells. The instrument further differentiates T lymphocytes frm non-T lymphocytes by measuring the green fluorescence signal of the OKT11-positive lymphocytes. In a typical sample, 1500--2500 lymphocytes are counted in 25 s. In a study of 158 patient samples, ranging from 1% to greater than 90% E-rosette-positive lymphocytes, the correlation coefficient between the manual E-rosette count and the flow immunofluorescence measurement is 0.943.
Automated intelligent microscopy (AIM) and its potential application in the clinical laboratory.
Authors: Deindoerfer F H , Boris W B , Gangwer J R , Laird C W , Tittsler J W
Published: January 2020

The microscope is the most ubiquitous instrument in the clinical laboratory. We discuss improvements in its use, in terms of "front-end" automation of specimen handling and "back-end" automation of image analysis: automated intelligent microscopy. Examples of spatial and spectral differentiation illustrate the potential of this automated version of microscopy as a useful tool with very powerful analytical capabilities for the clinical laboratory.
A computer-based maintenance reminder and record-keeping system for clinical laboratories.
Authors: Roberts B I , Mathews C L , Walton C J , Frazier G
Published: January 2020

"Maintenance" is all the activity an organization devotes to keeping instruments within performance specifications to assure accurate and precise operation. The increasing use of complex analytical instruments as "workhorses" in clinical laboratories requires more maintenance awareness by laboratory personnel. Record-keeping systems that document maintenance completion and that should prompt the continued performance of maintenance tasks have not kept up with instrumentation development. We report here a computer-based record-keeping and reminder system that lists weekly the maintenance items due for each work station in the laboratory, including the time required to complete each item. Written in BASIC, the system uses a DATABOSS data base management system running on a time-shared Digital Equipment Corporation PDP 11/60 computer with a RSTS V 7.0 operating system.
Analytical problems facing the development of electrochemical transducers for in vivo drug monitoring: an overview.
Authors: Pinkerton T C , Lawson B L
Published: January 2020

Even though significant progress has been made in the development of electrodes for the in vivo detection of catecholamines, oxygen, and various cations, there has been little research on the feasibility of continuously monitoring drugs in whole blood by electrochemical sensing devices. Electroanalytical problems associated with the development of such electrochemical transducers include the need for increased sensitivity and specificity, decreased biological matrix interferences, more rapid response times, improved miniaturization, and more reliable calibration procedures. We present a critical evaluation of potentiometric, conductometric, and amperometric techniques, with a brief review of basic principles and recent advances in ion-selective electrodes, chemical-sensing field-effect transistors, amperometric enzyme electrodes, and electrochemical immunosensors.
Use of monoclonal antibodies to detect human placental alkaline phosphatase.
Authors: De Groote G , De Waele P , Van de Voorde A , De Broe M , Fiers W
Published: January 2020

Convenient, sensitive, and specific solid-phase immunoassays involving monoclonal antibody are described for the determination of human placental alkaline phosphatase (hPLAP). An endogenous enzyme immunoassay combined the specificity of the immunological and the enzymatic reactions. Alternatively, a solid-phase "sandwich" radioimmunoassay involving immobilized polyclonal rabbit anti-hPLAP in combination with iodinated monoclonal antibody provided some additional advantages. Both tests can be used to detect hPLAP from various sources, e.g., in human sera during pregnancy or as a tumor marker. The radioimmunoassay detected an increase in hPLAP at nine weeks of gestation. We discuss the use of monoclonal antibodies for the differentiation of different alkaline phosphatase isoenzyme types by electrophoresis on starch gel.
Immunonephelometric determination of apolipoprotein A-1 in hyperlipoproteinemic serum.
Authors: Heuck C C , Erbe I , Flint-Hansen P
Published: January 2020

The intensity of scattered light varies with the size of the scattering particles. Studying the quantitative immunonephelometric determination of apolipoprotein A-1 (apo A-1), we observed that the different particle sizes of lipoproteins must be taken into account in immunonephelometry if apo A-1 is a constituent of the lipoprotein particles. Because interaction between large lipoproteins and immunocomplexes of high-density lipoproteins increases light scattering nonspecifically, false estimations may be obtained in serum with excessive hyperlipoproteinemia. Furthermore, the accessibility of antigenic sites of apo A-1 in intact high-density lipoproteins is limited. Immunonephelometry of apo A-1 in serum necessitates therefore the elimination of various interferents, which we have achieved by a single one-step extraction of lipids in a two-phase liquid system. With n-hexanol/polyfluoro-polychloro-polyethylene, about 90% of the serum lipids are extracted and only apolipoprotein B is precipitated at the interphase. This pretreatment eliminates the interferences caused by excessive hypertriglyceridemia and therefore greatly facilitates the endpoint immunonephelometry of apo A-1 in normal and pathological serum samples.