bluuebunny/crossref_metadata_2025 huggingface dataset:
Amylase from human serous ovarian tumors: purification and characterization.
Authors: Zakowski J J , Gregory M R , Bruns D E
Published: January 2020

Human serous-type ovarian tumors contain an acidic isoenzyme of amylase. Previous attempts at purification of tumor amylases have yielded preparations contaminated with other proteins. The purification scheme presented here incorporates an affinity-chromatography procedure, with use of cycloheptaamylose linked to epoxy-activated Sepharose, that is specific for alpha-amylase (EC 3.2.1.1). Purified amylase isoenzyme from a human serous ovarian tumor was characterized and compared with the purified salivary and pancreatic isoenzymes. All three were similar in amino acid composition, pH optimum, substrate specificity, calcium requirement, heat inactivation, and Km for maltotetraose substrate. The ovarian tumor amylase was similar to the salivary and distinct from the pancreatic enzyme by apparent molecular mass and doublet formation on sodium dodecyl sulfate--polyacrylamide electrophoresis, specific activity of pure enzyme, and sensitivity to specific alpha-amylase inhibitors. All three isoenzymes differed in net electrical charge as evidenced by diethylaminoethyl-Sephadex ion-exchange chromatography and isoelectric focusing. The tumor amylase is clearly distinct from the pancreatic and differs from the salivary enzyme in net electrical charge. Evidence is presented that this charge difference may reflect, at least in part, deamidation of an amylase that is similar to or identical with salivary amylase.
Noncompetitive enzyme-linked immunoassay for apolipoprotein B in serum.
Authors: Fievet C , Koffigan M , Ouvry D , Marcovina S , Moschetto Y , Fruchart J C
Published: January 2020

We used a noncompetitive enzyme-linked immunoassay to measure apolipoprotein B (apo-B) concentration in human plasma. Goat anti-lipoprotein B immunoglobulins were adsorbed to the surface of polystyrene balls. After washing, this solid-phase antibody was incubated with antigen (plasma from normal or hyperlipoproteinemic fasting subjects), washed, and then incubated with peroxidase-labeled goat anti-lipoprotein B IgG. After a last washing, we measured the bound label, which provided a direct measurement of the antigen. Under optimized assay conditions, the minimum detectable concentration was 50 ng per assay. The assay may be used to measure apo-B in different lipoprotein fractions (low- or very-low-density) and yields values that compared favorably with those obtained by electroimmunoassay (r = 0.86). The assay offers several advantages over existing techniques: sensitivity, specificity, simplicity, avoidance of radioisotopes, and potential for use with monoclonal antibodies.
On-line computer analysis of chemiluminescent reactions, with application to a luminescent immunoassay for free cortisol in urine.
Authors: Tommasi A , Pazzagli M , Damiani M , Salerno R , Messeri G , Magini A , Serio M
Published: January 2020

We interfaced a microcomputer on-line with a luminometer to acquire the light signal of chemiluminescent reactions from a photomultiplier and then compute significant parameters of light emission and kinetic "shape" indices. Using this system to study interferences from biological samples on the measurement of chemiluminescent reactions, we observed that such effects are usually associated with modifications of the shape of the light-emission kinetics. These results suggest that a simultaneous evaluation of the shape of a chemiluminescent reaction and the measurement of light emission can be combined to assess luminescent immunoassays as an internal control of the interferences in measurements of the chemiluminescent tracer. As an example of this approach, we developed and validated a luminescent immunoassay for free cortisol in diluted urine. Dextran-coated charcoal is used for bound-free separation.
Determination of polyols in serum by selected ion monitoring.
Authors: Roboz J , Kappatos D C , Greaves J , Holland J F
Published: January 2020

We used gas chromatography-chemical ionization mass spectrometry with selected ion monitoring of the (M-59)+ ions to determine polyols (as their peracetyl derivatives) in serum. The internal standard was 2-deoxygalactitol. Mean (and SD) polyol concentrations (mg/L) in 33 normal sera were: erythritol 0.45 (0.14), threitol 0.20 (0.06), adonitol 0.06 (0.02), arabinitol 0.37 (0.12), xylitol 0.05 (0.02), mannitol 0.41 (0.45), galactitol 0.15 (0.11), sorbitol 0.16 (0.11). Arabinitol determined in the same 33 normal sera by trimethylsilylation was 0.39 (0.13) mg/L. In 32 samples from cancer patients with normal creatinine, all polyol concentrations were in the normal range except for seven increased erythritol and one increased threitol concentrations. In all six patients with renal dysfunction we found increased erythritol and arabinitol, and increased threitol and mannitol in four of the six. Initial results showed that only arabinitol concentrations increased in invasive candidiasis.
Sampling and analysis techniques used in a blood lead survey of 1241 children in Port Pirie, South Australia.
Authors: Sinclair D F , Dohnt B R
Published: January 2020

Methods are described for taking finger-prick blood samples and for analyzing these samples for lead. Quality-control techniques and procedures for the validation of all results are described. Results of a survey of 1241 children are given and results for 149 venous samples are compared with results for the corresponding finger-prick samples. This finger-prick sampling method evidently is suitable for use in screening large numbers of children.
Reaction of picrate with creatinine and cepha antibiotics.
Authors: Kroll M H , Hagengruber C , Elin R J
Published: January 2020

The concentration of creatinine in serum, which is used to estimate glomerular filtration rate, is measured by reaction with alkaline picrate, but this reaction is not specific for creatinine. Although several other cephalosporin antibiotics have been reported not to react with picrate, we reacted picrate with creatinine, cefoxitin, penicillin, and eight different cephalosporins, and found that all compounds reacted with picrate and showed superimposable spectrophotograms with absorption maxima at 485 nm. From these results we conclude that the color-absorbing moiety of the product is the picrate molecule. Further, the structure common to creatinine and the cephalosporins, cefoxitin, or penicillin is the carbonyl group attached to a nitrogen and a carbon atom. We postulate that the carbonyl group with the adjacent carbon and nitrogen atoms is probably the chemical moiety that reacts with picrate to absorb energy at 485 nm.
Simple colorimetry of glycated serum protein in a centrifugal analyzer.
Authors: Lloyd D , Marples J
Published: January 2020

A recently described (Clin Chim Acta 127: 87-95, 1982) colorimetric assay for glycated proteins in serum exploits their ketoamine activity to reduce 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)bis [2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (nitroblue tetrazolium) in alkaline solution; the authors termed this the "fructosamine assay." The method is simple, requiring only the addition of one reagent and measurement of the absorbance change during 5 min; results are expressed relative to a synthetic standard. We have adapted this for use in a centrifugal analyzer and report its performance, both analytically and as an index of hyperglycemia. Precision is good (between-batch CV 2.1%), the reagent is stable and inexpensive, and the procedure is rapid (75 samples per hour). Albumin influences the measurement, but for concentrations greater than or equal to 35 g/L this was not a serious problem. Normal and diabetic populations can be clearly discriminated (p less than 0.001). The test detected 25 (84%) of the 30 untreated diabetics studied and gave four false positives (8%). The results correlate well with those for glucose in plasma of fasting subjects (r = 0.87) and for hemoglobin A1 (r = 0.80).
A rapid silver-stain procedure for use with routine electrophoresis of cerebrospinal fluid on agarose gels.
Authors: Lubahn D B , Silverman L M
Published: January 2020

Oligoclonal banding patterns, when present in cerebrospinal fluid (CSF) electrophoresed on agarose gels, are important in the diagnosis of multiple sclerosis and other demyelinating diseases. Typically, an 80-fold concentration of 2.5 mL of CSF has been needed before the oligoclonal banding pattern could be detected with Coomassie Brilliant Blue stain. This excessive volume requirement often is a problem, particularly in children. We describe here an improved silver-stain procedure with which we routinely can detect the oligoclonal banding pattern in 1 to 3 microL of unconcentrated CSF. The procedure is inexpensive and takes less than 45 min. Our method is quick, safe, sensitive, and easily performed in the clinical laboratory.
Modified radioassay for measuring asialoglycoprotein in serum.
Authors: Byrn R , Thomas P , Medrek P , Spigelman Z , Zamcheck N
Published: January 2020

Asialoglycoproteins are removed from the circulation by the carbohydrate-specific hepatic binding protein in the rat. Asialoglycoprotein in human serum can be detected by an inhibition assay in which the binding of 125I-labeled asialo-alpha 1-acid glycoprotein (ASAG) to purified hepatocyte membranes is inhibited by known amounts of ASAG or patient's serum. We were able to reproducibly measure inhibition equivalent to that of 1 to 20 ng of ASAG. The mean concentration of inhibitor in 11 healthy control patients was equivalent to 0.153 (SD = 0.051) mg of ASAG per liter, measured in 20 microL of serum. Significant increases of inhibitor were observed in patients with malignant extrahepatic biliary obstruction, alcoholic liver disease, viral hepatitis, or liver metastases, but not in those with benign biliary obstruction. For a set of six control sera the intra- and interassay CVs were 9.1% and 25.2%, respectively (n = 3 each).
Analyses for progesterone in serum by gas chromatography/mass spectrometry: target data for external quality assessment of routine assays.
Authors: Gaskell S J , Brownsey B G , Groom G V
Published: January 2020

We describe a procedure for measuring progesterone in plasma and serum by isotope dilution and mass spectrometry. Extraction with use of a microcellulose-coupled antiserum is followed by conversion to the 3-enol heptafluorobutyrate and analysis by gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring, at a resolution of 5000. Interassay CVs were 1.5 to 5.4% for the concentration range 13 to 43 nmol/L. Analyses of various serum volumes showed excellent linearity. Accurate determination of progesterone added to serum was demonstrated. Plasma and serum pools were analyzed to provide target data for use in the U.K. national external quality-assessment scheme for progesterone assays. Direct, non-extraction radioimmunoassays and those incorporating solvent extraction both showed a positive bias with respect to data obtained by the present procedure, but the bias was more marked with the direct assays.
Vanadium in human serum, as determined by neutron activation analysis.
Authors: Simonoff M , Llabador Y , Peers A M , Simonoff G N
Published: January 2020

Vanadium concentrations have been measured in the serum of 23 healthy subjects by neutron activation analysis, with post-irradiation chemical separation. The values obtained fall in a fairly narrow range (260-1300 ng/L, mean 670 ng/L), which suggests a physiological role for this element. We checked the method by analysis of standard (U.S. National Bureau of Standards) water samples and serum samples supplemented with 48V, and we found excellent agreement with expected results in both cases. We consider the reported results for human serum to be more reliable than those obtained by atomic absorption spectroscopy.
Calculated vs measured plasma osmolalities revisited.
Authors: Bhagat C I , Garcia-Webb P , Fletcher E , Beilby J P
Published: January 2020

The osmolalities of 100 plasma samples were measured and compared with the osmolalities calculated from the plasma concentrations (mmol/L) of sodium, potassium, glucose, and urea by several different formulae. The formula recommended by Dorwart and Chalmers (Clin Chem 21: 190, 1975) gave inferior results to those obtained with our "most accurate" formula: osmolality = 1.89 Na + 1.38 K + 1.03 urea + 1.08 glucose + 7.45. We recommend using this formula for calculation of osmolality on equipment linked to a computer. However, for simplicity, and to reduce the possibility of calculation errors, the following formula can be used for manual calculations: osmolality = 1.86 (Na + K) + glucose + urea + 10.
The Quantab strip in the measurement of urinary chloride and sodium concentrations.
Authors: Sloan P J , Beevers G , Baxter F E
Published: January 2020

The "Quantab" strip (Ames) measures chloride in fluids. For sodium chloride solutions and urine we found very good correlations between the Quantab reading and the chloride concentration as measured by chemical assay (r = 0.95 for chloride and r = 0.85 for sodium in urine). The strip gave reproducible results over the temperature range 4 to 37 degrees C. There was very little inter- and intra-observer variation in reading the strip. Although 10 to 23 min is required to complete the reaction, the strip reading is stable thereafter. We suggest that the strip could be useful in epidemiological studies of urinary sodium concentration and clinically in helping patients adhere to a low-salt diet.
Two direct (nonchromatographic) assays for 25-hydroxyvitamin D.
Authors: Bouillon R , Van Herck E , Jans I , Tan B K , Van Baelen H , De Moor P
Published: January 2020

We describe direct, nonchromatographic assays for 25-hydroxyvitamin D3 in which we use either rat vitamin D-binding protein or rabbit antibodies to 25-hydroxyvitamin D3-3-hemisuccinate-bovine serum albumin as binding protein. Nonspecific interferences in serum could be eliminated by an appropriate extraction method or in obtaining data for the calibration curve, by using vitamin D-free human serum. The latter is prepared by affinity chromatography with use of immobilized antibodies against human vitamin D-binding protein. Values obtained by the direct assays and those obtained by two different chromatographic methods correlated well (r greater than 0.95). The direct competitive protein-binding assay overestimated the true 25-hydroxyvitamin D3 concentration by about 20%, but this percentage was constant from 5 to 600 micrograms/L. Overestimation by the direct radioimmunoassay was less than 10%. These two direct assays for 25-hydroxyvitamin D3 allow reliable, rapid, and simple screening for vitamin D deficiency or excess.
Biological variance of total lactate dehydrogenase and its isoenzymes in human serum.
Authors: Moses G C , Henderson A R
Published: January 2020

We measured the variance components of total lactate dehydrogenase (LD; EC 1.1.1.27) activity and isoenzymes in sera of 24 healthy laboratory staff, ages 23-50 years, over a 12-month period. We used the first six weeks' results to establish baseline values for each analyte in each individual. These values were "normally" distributed for total LD and isoenzymes 1 through 4, but values for isoenzyme-5 were skewed slightly to the left. The overall means and variances determined for the remaining 10 months were not significantly (p greater than 0.05) different from the corresponding baseline values. Average variances (SD2) for the longer-term (10-month) study were: intra-individual, 732, 1.92, 2.25, 1.09, 1.11, and 2.25, and inter-individual, 1988, 5.03, 1.76, 1.57, 1.21, and 2.01 for total LD (U/L) and LD-1 through LD-5 (% of total), respectively. Average long-term analytical variability was less than 35% of the total variance for the five isoenzymes and 6% for total LD, and was characteristic of the individual. There were no significant differences in mean activities with respect to age or sex. All six analytes exhibited slight seasonal variations.
An automated "high-pressure" liquid-chromatographic assay for hemoglobin A1c.
Authors: Ellis G , Diamandis E P , Giesbrecht E E , Daneman D , Allen L C
Published: January 2020

An automated "high-pressure" liquid-chromatographic assay for hemoglobin A1c is described. We use a 45-min incubation in acetate buffer (pH 5.5) to eliminate labile glycated hemoglobins. In this automated system conventional modules are used but it incorporates a solvent-switching valve to select either of two buffers, which differ in pH and NaCl concentration. The chromatographic column contains "polyCAT" (a weak cation-exchanger, polyaspartic acid linked to silica). Run time is 14 min per sample. The method is precise and results correlate well with those by other ion-exchange procedures.
New photometric assay for chymotrypsin in stool.
Authors: Kaspar P , Möller G , Wahlefeld A
Published: January 2020

In this new photometric assay fecal samples are pretreated with detergent and high concentrations of salts. The subsequent kinetic enzyme determination step involves the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-4-nitroanilide. The sample pretreatment assures a nearly complete solubilization of the formerly particle-bound enzyme, thus permitting determination of the enzyme activity in either the suspension or the supernate after centrifugation. Furthermore this pretreatment enhances the enzyme activity and decreases the Km value. Results by this assay correlate well with those by classical titrimetry and the method is easily adapted to automated systems.
Kinetic approach for the enzymic determination of creatinine.
Authors: Perakis N , Wolff C M
Published: January 2020

By exploiting the kinetics of NADH consumption in the enzyme system creatinine amidohydrolase + creatine kinase + pyruvate kinase + lactate dehydrogenase, one can determine creatinine in serum in the range of 1 to 90 mg/L. By using the conditions defined here, this determination can be made with a single measurement of the rate of disappearance of NADH. The analytical rate measurement is made at a fixed time (2 min) after the sample is introduced into the enzyme solution. For this fixed time, the interferences of creatine and pyruvate are eliminated. Results so obtained for creatinine in human blood serum samples correlated well (r = 0.98) with those obtained by the classical Jaffé-reaction method. Run-to-run reproducibility (CV) was 3%, and the limit of detection was 1 mg/L.
Five methods for determining low-density lipoprotein cholesterol compared.
Authors: Demacker P N , Hijmans A G , Brenninkmeijer B J , Jansen A P , van 't Laar A
Published: January 2020

We evaluated three precipitation methods for determination of low-density lipoprotein cholesterol in serum and an indirect method involving the Friedewald formula (Clin Chem 18: 499-502, 1972) by comparison with results by ultracentrifugation. The results of all methods for 83 sera, including 59 hyperlipidemic type IIA, IIB, and IV sera agreed very well, at least for concentrations of serum triglycerides below 8 mmol/L. The accuracy of the Friedewald formula was confirmed in 285 other sera, including 66 sera with triglycerides content between 4.52 and 8.0 mmol/L. For type III sera, the precipitation methods produced similar values to those obtained with the Friedewald formula, all being much higher than the ultracentrifugation values. Density-gradient ultracentrifugation showed that the very-low-density lipoprotein remnants in type III sera almost completely coprecipitated with the low-density lipoproteins. The precipitation methods are not only accurate but also very precise (CV less than 5%); they can therefore be used in clinical laboratories to measure atherogenic low-density lipoproteins plus the remnants of very-low-density lipoproteins. However, when serum triglycerides and high-density lipoprotein cholesterol also are determined, the Friedewald formula is a reliable alternative.
Unreliability of immunochemical determination of lactate dehydrogenase isoenzyme-1 in heparinized plasma.
Authors: Lum G
Published: January 2020

Paired serum and heparinized plasma samples were assayed simultaneously for lactate dehydrogenase (EC 1.1.1.27) isoenzyme 1 (LD1) activity by a commercially available immunochemical procedure. For all sera specimens tested, only LD1 activity was detected. For heparinized plasma, random discrepancies in LD1 activity were noted at normal (Group I), borderline (Group II), and increased (Group III) total LD activity. Incomplete precipitation of LD-M subunits was confirmed by electrophoresis and occurred in eight of 15, four of 13, and eight of 22 instances (total: 20/50, or 40%) with a mean difference of 13, 10.6, and 18.2 U/L (8.3, 4.0, and 4.4%) in Groups I, II, and III, respectively. We conclude that heparinized plasma is an unsuitable sample for the immunochemical determination of LD1.
Inadequate hemolysis of erythrocytes on reagent strips at low pH causes false-negative readings.
Authors: Levin K , Engström I
Published: January 2020

Patients' urines gave an unexpectedly high percentage of false-negative results for erythrocytes on a reagent strip, even when the test field for ascorbic acid indicated that none of this substance was present to inhibit the test reaction. By adding known amounts of erythrocytes to normal urines, we found that this phenomenon was time-dependent and occurred with three different brands of reagent strips in urines having a pH below 6.0. Hemolysis of erythrocytes was totally inhibited below pH 5.1 after 4 h of incubation with 50 X 10(6) erythrocytes per liter. The inhibition is apparently caused by shrinkage of the erythrocytes, rending them insensitive to the hemolytic agents in the different reagent strips tested.
Identification of polypeptides on two-dimensional electrophoresis gels by amino acid composition.
Authors: Latter G I , Burbeck S , Fleming J , Leavitt J
Published: January 2020

We present a method that can, in principle, provide tentative identification of a substantial proportion of the polypeptides resolvable on a given two-dimensional electrophoresis gel. Computerized microdensitometry of autoradiograms from 20 gels labeled in turn with each of the 20 common amino acids provides the data for simultaneously measuring the amino acid composition of all polypeptides of interest on the gel. These compositions are then compared with computer data bases of known protein compositions. Similarity between a known and an unknown polypeptide with comparable molecular mass indicates a potential identification, which can then be confirmed with conventional techniques. We illustrate this technique by applying it to the identification of proteins in a transformed human cell line (HuT-14).
"Ultrasensitive" silver stains: their use exemplified in the study of normal human cerebrospinal fluid proteins separated by two-dimensional electrophoresis.
Authors: Merril C R , Harrington M G
Published: January 2020

We have developed a series of photochemical silver stains that are more than 100 times as sensitive as the Coomassie Blue stains. These stains facilitate the quantitative estimation of proteins in two-dimensional gel patterns. Recently, we discovered that by using light (photodevelopment) to catalyze the reduction of ionic silver to metallic silver, we could produce an image of proteins on polyacrylamide gels within 10 min after their electrophoretic separation. We used these stains to detect proteins resolved in two-dimensional electrophoretograms of lumbar cerebrospinal fluid from 51 normal human volunteers, finding no qualitative protein changes in the more than 300 CSF polypeptides resolved on each individual's electrophoretogram. We quantified 67 of these proteins per individual with computer-assisted densitometry, only five of which showed significant quantitative age-related alterations. We saw no sex-related differences. This consistency in the protein pattern for cerebrospinal fluid from normal individuals allows it to serve as a baseline for comparisons with disease states.
Two-dimensional electrophoresis of proteins in human serum: improved resolution by use of narrow pH gradients and prolonged electrophoresis.
Authors: Marshall T , Williams K M , Vesterberg O
Published: January 2020

The limited resolution of serum proteins achieved with a simplified technique of two-dimensional electrophoresis (Clin. Chim. Acta 103: 51-59, 1980) has been improved by using different ampholyte (Ampholine) mixtures in the first dimension, to obtain relatively shallow pH gradients, and prolonged electrophoresis time in the second dimension. The technique has been further simplified, without negative effect, by decreasing the concentration of non-ionic detergent (first dimension), omitting both sodium dodecyl sulfate equilibration and the use of a stacking gel (second dimension), and by using an improved silver-staining procedure. Reverse-polarity isoelectric focusing and non-equilibrium pH-gradient electrophoresis have been successfully used to resolve the basic polypeptides in serum after deleting sodium dodecyl sulfate from the sample preparation. These combined techniques reveal over 1100 polypeptides in human serum. After immunodeletion of albumin, additional serum polypeptides are seen. Immunodeletion of serum proteins from plasma reveals polypeptides that are relatively specific to plasma.
Changes in the expression of hepatocyte protein gene-products associated with adaptation of cells to primary culture.
Authors: Colbert R A , Amatruda J M , Young D A
Published: January 2020

Expression of hepatocyte protein gene-products changes as cells adapt to the environment of tissue culture. We examined such changes, using "giant" two-dimensional (2-D) gel electrophoresis. Over 80 cellular and secreted proteins, about 3% of the 2500 we examined, were found to change spontaneously during the 20-h culture period. The magnitude of these changes was estimated to range from 1.5- to nearly 100-fold. If dexamethasone is included in the culture medium, the overall production of secreted proteins is maintained, but in addition it exerts major influences on the expression of individual protein gene-products. Ten of the spontaneous changes are enhanced and eight are unaffected, but most are substantially retarded (32) or even reversed (27). There are also large inductions or repressions in 12 proteins that do not change spontaneously in culture. We have tentatively identified tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, gamma-fibrinogen, and alpha 1-acid glycoprotein to be among those induced by dexamethasone. Evidently, many more changes occur as these cells adapt to tissue culture than was previously appreciated. These results emphasize the power of 2-D gel analysis in monitoring the protein phenotype of cells. We have also shown that glucocorticoids are important in maintaining the overall synthesis of secreted proteins and the protein phenotype of isolated hepatocytes in primary culture.
Leukocyte membrane proteins in chronic lymphocytic leukemia, as studied by two-dimensional gel electrophoresis.
Authors: Willard-Gallo K E , Humblet Y , Symann M
Published: January 2020

We evaluated protein expression in leukocytes from 20 patients with chronic lymphocytic leukemia (CLL), including one with the rare T-cell form of the disease. To identify proteins that potentially could be used to characterize leukemia or as candidates for new markers of differentiation, we studied cell and membrane extracts from these leukemic cells. We used immune precipitation and extraction of integral membrane proteins with Triton X-114 to identify known proteins on the surface of these cells. Extraction with Triton X-114 in the presence of protease inhibitors yielded reproducible membrane extracts, which we examined by two-dimensional gel electrophoresis. Of the approximately 2000 proteins or protein subunits so resolved from cell lysates and the 450 from membrane extracts of leukocytes from patients with T- and B-cell CLL, we were able to identify spots corresponding to the proteins designated by the OKT.4 and OKT.10 antibodies, the human class I and II histocompatibility antigens, beta 2-microglobulin, and surface IgM. We also defined sets of proteins that are characteristically expressed on the membranes of leukemic T or B cells, some of which correspond to previously defined markers of normal leukocyte subpopulations.
Two-dimensional electrophoresis of human plasma apolipoproteins.
Authors: Sprecher D L , Taam L , Brewer H B
Published: January 2020

The two-dimensional electrophoretic method with silver staining we describe better resolves plasma apolipoproteins (apo) than any procedure previously described. It can be used to screen for abnormalities in apoA-I, apoA-II, apoA-IV, apoC-II, apoC-III, apoD, apoE, and apoH. In addition, this is the first presentation of apoD and apoH on two-dimensional gels. This electrophoretic method will permit the quantification of plasma apolipoproteins by computer imaging. Such detailed analysis of the plasma apolipoproteins should provide a better understanding of apolipoprotein function and the role these apolipoproteins play in lipoprotein metabolism and the pathophysiology of dyslipoproteinemias and coronary artery disease.
Pouring reproducible gradients in gels under computer control: new devices for simultaneous delivery of two independent gradients, for more flexible slope and pH range of immobilized pH gradients.
Authors: Altland K , Altland A
Published: January 2020

Recently we described (Electrophoresis 5: 143-147, 1984) a new device for producing density and solute gradients, involving computer-controlled coöperation of stepmotor-driven high-precision burettes, the purpose being to substantially improve reproducibility, flexibility, and documentation of gradients used in gels and in other applications in biochemistry. Here we present the functional principle of three modified and partly alternative devices, based on simultaneous delivery of a density gradient and a non-density gradient. They provide unlimited flexibility of choice for the slope of the non-density gradient, which is stabilized by the density gradient. They seem especially useful for pouring immobilized pH gradients in gels of wide pH ranges (between pH 3.5 and 10) where localized flattening of the gradient is needed to selectively improve resolution. Used in two-dimensional electrophoretic analysis, they should considerably improve control of spot coördinates, standardization of the technique, and interlaboratory data exchange.
Direct solid-phase fluoroenzymeimmunoassay of 5 beta-pregnane-3 alpha, 20 alpha-diol-3 alpha-glucuronide in urine.
Authors: Shah H , Saranko A M , Härkönen M , Adlercreutz H
Published: January 2020

A competitive solid-phase fluoroenzymeimmunoassay has been developed for rapidly measuring 5 beta-pregnane-3 alpha, 20 alpha-diol-3 alpha-glucuronide (Pd-3G) directly in diluted specimens of pre-breakfast urine. The assay involves use of an antiserum to Pd-3G and enzyme-labeled antigen prepared by chemically linking glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to Pd-3G. Antibody-bound and free antigen are separated by use of a solid-phase double antibody: sheep anti-rabbit gamma-globulin coupled to cellulose particles. The solid phase, isolated by centrifugation, is washed free of labeled antigen and endogenous enzyme interferences. Enzyme activity in the bound fraction is then measured fluorometrically with glucose 6-phosphate as the substrate and generated NADPH as a fluorogenic indicator of enzyme activity. The assay is sufficiently sensitive (30-35 pg per assay tube), specific, and reliable for routine use, and results correlate well (r = 0.98) with those by an established specific radioimmunoassay. Because it is suited to routine use, this assay may be applied to detecting ovulation, assessing the function of corpus luteum, and monitoring early pregnancy.
Continuous-flow determination of primary bile acids, by bioluminescence, with use of nylon-immobilized bacterial enzymes.
Authors: Roda A , Girotti S , Ghini S , Grigolo B , Carrea G , Bovara R
Published: January 2020

We describe a continuous-flow bioluminescence method for measuring primary bile acid in serum, with nylon as the solid support. 7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1. 159), a bacterial luciferase (EC 1.14.14.3), and NAD+:FMN oxido-reductase (EC 1.6.8.1) are covalently co-immobilized on a nylon coil (1 m X 1.0 mm i.d.). The assay is highly specific for 7 alpha-hydroxy bile acids. Other bile acids and steroids do not interfere. The continuous-flow light-emitting system, in which the reactor (nylon coil) is placed in front of a photomultiplier tube inside a luminometer, is versatile and simple. The flow is air-segmented, and serum samples (5-50 microL) can be injected directly. Concentration and response are linearly related from 10 to 2500 pmol per assay tube. The precision of the method is satisfactory (CV 5-10%), both inter- and intra-assay. We validated the technique by comparing results with those by RIA, enzyme immunoassay, and "high-performance" liquid chromatography. More than 20 samples an hour can be analyzed, with no carryover. The nylon-immobilized enzymes are stable for more than two months, and greater than 500 samples can be analyzed with use of a few milligrams of enzymes. Normal values for bile acid content of serum ranged from 1 to 2.5 mumol/L, in agreement with those obtained by other methods.
Enzyme-linked immunosorbent microassay and hemagglutination compared for detection of thyroglobulin and thyroid microsomal autoantibodies.
Authors: Roman S H , Korn F , Davies T F
Published: January 2020

We have evaluated for their potential use in the routine clinical laboratory enzyme-linked immunosorbent assays (ELISA) for human thyroglobulin antibodies (hTg-Ab) and microsomal antibodies (M-Ab). Results are expressed in terms of an "ELISA Index," based on comparison with a laboratory standard. The specificity of both ELISA assays is shown by dose-dependent inhibition of the hTg-Ab and M-Ab activities of the laboratory standards by the appropriate specific antigens. Similar concentrations of ovalbumin had no significant effect on the standard activity in both assays. For consecutive samples evaluated for hTg-Ab (n = 113) and M-Ab (n = 106) by ELISA and hemagglutination, rank order analysis of the results showed a highly significant correlation between the methods (r = 0.81, p = less than 0.001 for hTg-Ab; r = 0.82, p = less than 0.001 for M-Ab). However, 8/47 (17%) of samples positive in the hTg-Ab ELISA were negative by hemagglutination, and 7/69 (12%) of samples positive in the M-Ab ELISA were negative by hemagglutination. We effectively excluded the possibility of false positivity of these specimens by ELISA by blocking specimen positivity with the specific antigens in 12 of 14 specimens investigated. We conclude that ELISA techniques for human thyroid autoantibodies are sensitive and specific, easy to initiate, objective, and capable of use in large-scale screening. They are superior to standard hemagglutination techniques by having an increased detection rate for hTg-Ab and M-Ab.
Characterization of apparent lactate dehydrogenase isoenzyme 6: a lactate-independent dehydrogenase.
Authors: Podlasek S J , McPherson R A , Threatte G A
Published: January 2020

We found in a prospective study of lactate dehydrogenase isoenzyme 6 (LD6) in human tissues obtained at autopsy that LD6 was usually present in liver when death was preceded by prolonged hypotension or impaired ventilation but not in cases of sudden death. Other organs containing LD6 were kidney and spleen. LD6 is heat stable, differs from H-subunit-containing LD isoenzymes by pyruvate resistance, differs from M-subunit-containing LD isoenzymes by immunoprecipitation, and is distinct from spermatic LDX. LD6 from liver extracts acted without lactate as substrate and could be enhanced by ethanol added to the substrate. These results indicate that LD6 is not a true lactate dehydrogenase, and that it frequently appears in severe liver injury.
Methods for extracting phospholipids from human amniotic fluid compared.
Authors: Duck-Chong C G , Baker G J , Murdoch S R , Price R M
Published: January 2020

Phospholipids were extracted from human amniotic fluid by various procedures, including the two most commonly applied to amniotic fluid for evaluation of fetal lung maturity. We find that the yield of phospholipid is greatly procedure dependent. This should be taken into account when one is considering the various reported methods of evaluating fetal lung maturity, because in some of them phospholipid data are expressed in terms of absolute concentration in the amniotic fluid. There were also significant differences in phospholipid composition in extracts prepared by the various procedures, but in general these were not large enough to influence evaluation of fetal lung maturity by methods in which phospholipid data are expressed in relative terms, as ratios or percentages--e.g., in the lecithin/sphingomyelin ratio and "lung profile" procedures. In the extraction method originally recommended for determination of the lecithin/sphingomyelin ratio, both the yield and composition of phospholipid depend on the extent of mixing.
Rapid quantification of pentobarbital in serum by fluorescence polarization immunoassay.
Authors: Li P K , Lee J T , Schreiber R M
Published: January 2020

The commercially available fluorescence polarization immunoasay (FPIA) method for phenobarbital is demonstrated to be useful in the quantification of pentobarbital in serum. The effective range for the method is 1.0 to 80.0 mg/L. The method is simple, precise, and fast. The application of the method is limited to patients receiving pentobarbital as the only barbiturate. The FPIA is recommended for use in clinical laboratories when a rapid estimation of pentobarbital is required in monitoring patients undergoing high-dose pentobarbital therapy.
Interconversion and comparison of the results of three methods for cholinesterase in serum.
Authors: Newman M A , Que Hee S S
Published: January 2020

We investigated three methods for determination of cholinesterase (ChE) in human blood sera, using reference sera, sera of known ChE content, and serum variously diluted with deactivated sera. The methods were the Du Pont aca analyzer, the Pfizer "ChE-tel" method, and the Michel method with and without direct reagent-blank correction. With respect to precision and reproducibility the methods ranked: aca greater than Michel method corrected directly for reagent pH changes (Michel) greater than ChE-tel method greater than Michel method corrected indirectly for reagent pH changes. The interrelationships derived, in terms of each method's individual units were: Michel = (0.054 +/- 0.001) aca - (0.043 +/- 0.086), r2 = 0.947 ChE-tel = (4.44 +/- 0.30) aca - (5.11 +/- 0.99), r2 = 0.959 ChE-tel = (80.2 +/- 5.8) Michel + (0.028 +/- 0.240), r2 = 0.947 The ChE-tel method was subject to variations due to the quality of different kits. The aca results were validated by a collaborative laboratory study. Sera of known activity can be used effectively for accurate calibration.
Adaptation of EMIT reagents to the Cobas Bio centrifugal analyzer.
Authors: Hamlin C R , Sullivan P A
Published: January 2020

Our adaptation of EMIT enzyme immunoassay reagents to the Cobas Bio centrifugal analyzer avoids the decrease in precision at the upper end of the therapeutic range reported in other adaptations. Long-term (12-month) CVs for lidocaine, valproic acid, and tobramycin were 5% or less; for quinidine, acetaminophen, and phenytoin, long-term CVs ranged from 7.2% to 8.6%.
Improved liquid-chromatographic determination of mexiletine, an antiarrhythmic drug, in plasma.
Authors: Mastropaolo W , Holmes D R , Osborn M J , Rooke J , Moyer T P
Published: January 2020

In this improved reversed-phase liquid-chromatographic procedure for determination of mexiletine in plasma, mexiletine and an internal standard, chlorodisopyramide, are extracted with methylene chloride from 0.5 mL of serum or plasma; the extract is then concentrated and injected onto a C18 chromatographic column. Mexiletine in the column effluent is detected by monitoring absorbance at 210 nm. It is quantified by use of mexiletine-internal standard peak-height ratios. The relation between this ratio and mexiletine concentration is linear from 0.1 to 5.0 mg/L. The lower limit of detection is about 50 micrograms/L. At a mexiletine concentration of 2.0 mg/L in serum, intrarun precision (CV) is 2.9% and inter-run precision is 5.9%; at 0.5 mg/L, these CVs are 5.7% and 9.6%, respectively. Analytical recovery of added mexiletine in serum is 68-88%. Therapeutic concentrations of some commonly administered drugs in patients' specimens did not interfere. In serum from 38 patients receiving mexiletine for cardiac arrhythmia, concentrations measured by this method correlated with therapeutic efficacy.
Hyponatremia: a diagnostic problem.
Authors: Walmsley R N , White G H
Published: January 2020

Hyponatremia, a common clinical problem, may result from a wide variety of causes. Treatment can vary from fluid restriction to rehydration, depending on the pathogenesis of the disorder. Essential to evaluation of hyponatremic patients is correct estimation of the status of the extracellular fluid volume, because this determines the type of therapy: a patient who is hypovolemic will not benefit from fluid restriction; a euvolemic patient should not be "rehydrated." To distinguish between these two conditions requires a searching history and clinical examination, including measurement of the blood pressure both while the subject is standing and lying. Not all patients with the classical biochemical features of the syndrome of inappropriate secretion of vasopressin (low plasma [Na+] and osmolality, high urinary [Na+] and osmolality) will have the disorder, because these same features may also be present in other hyponatremic disorders, including hypovolemic hyponatremia.
Colorimetry of hemoglobin in plasma with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).
Authors: Takayanagi M , Yashiro T
Published: January 2020

Hemoglobin in plasma can be determined by the color-developing action of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid), which is oxidized to a colored form by a peroxidase-like effect of hemoglobin in the presence of hydrogen peroxide. Sensitivity, precision, and accuracy are discussed. The calibration curve is linear for hemoglobin concentrations up to 1 g/L; the minimum detectable concentration is 20 mg/L. The within-run precision (CV) was 2.39%, analytical recovery 101.8%. Interference from plasma proteins and lipids was eliminated by centrifuging the reaction mixture before measuring its absorbance at 410 nm.
Normal reference-intervals for 20 biochemical variables in healthy infants, children, and adolescents.
Authors: Gómez P , Coca C , Vargas C , Acebillo J , Martinez A
Published: January 2020

We determined normal reference values from data on sera of 2099 outpatient children (ages one week to 14 years) at our institution. Using a continuous-flow instrument (SMAC, Technicon), we determined the following analytes in each serum sample: glucose, creatinine, uric acid, inorganic phosphorus, sodium, potassium, chloride, total CO2, iron, cholesterol, triglycerides, total protein, albumin, total bilirubin, creatine kinase, lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and calcium. The resulting data were coded and subsequently processed in an IBM 370 computer, and the reference values (3rd and 97th percentiles) were defined for each analyte. A two-way analysis of variance was also done to determine the influence of age and sex on results of these 20 biochemical tests.
Quantification of nonenzymically glycated albumin and total serum protein by affinity chromatography.
Authors: Yatscoff R W , Tevaarwerk G J , MacDonald J C
Published: January 2020

We have evaluated an affinity-chromatographic procedure for determination of glycated albumin (GA) and glycated total serum protein (GSP). Recovery of these analytes was inversely related to free glucose concentration, thus necessitating removal of free glucose. For this we used molecular-exclusion chromatography on G-25 Sephadex, or dialysis, the latter procedure resulting in significantly (p less than 0.05) lower concentrations of GSP and GA. Total protein concentration and percent glycation are also inversely related, and so protein concentrations must be standardized before the assay. Within- and between-run CVs for both GSP and GA were less than 6.5 and 18%, respectively, the determination of GA being generally the more precise of the two. Labile glycated fractions, lipemia, icterus, hemolysis, and type of anticoagulant did not affect the results, but assay temperature did. Diabetic subjects showed substantially higher concentrations of GA and GSP than did normal subjects. Because of the life span of these analytes in circulation, their measurement may provide a short-term index of glycemic control.
Some factors affecting determination of carotenoids in serum.
Authors: Mathews-Roth M M , Stampfer M J
Published: January 2020

Because of increasing interest in carotenoid pigments, we conducted a study of the methods of determining carotenoids in serum. We found 1 mol of KOH per liter of absolute methanol to be the most effective saponifying solution. The absorbance of beta-carotene in petroleum ether, the extraction solvent, is proportional to dilution up to an absorbance of 0.85 at 450 nm. Beta-carotene in petroleum ether solution is not impracticably sensitive to ambient light at room temperature. However, if vitamin A is also to be measured in these serum specimens or petroleum ether extracts, exposure to light should be minimized. We found that serum may be shipped either in cold packs or at ambient temperature (tested up to five days) without significant change in carotenoid concentration. Serum samples for carotenoid determination are best stored at -70 degrees C: samples stored at -20 degrees C deteriorate substantially over several months.
Liquid-chromatographic determination of eight tri- and tetracyclic antidepressants and their major active metabolites.
Authors: Sutfin T A , D'Ambrosio R , Jusko W J
Published: January 2020

A "high-performance" liquid-chromatographic method is presented for monitoring the therapeutic concentrations, in plasma, of eight tri- and tetracyclic antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, doxepin, clomipramine, maprotiline, and protriptyline) and their major active metabolites. One of two internal standards is added to 0.5 mL of drug-containing plasma, the pH is adjusted to about 9.5 with borate buffer, and the sample is extracted with isoamyl alcohol in hexane. The extracts are chromatographed on a column of silica and absorbance of the effluent is measured at either 214 or 254 nm. Chromatographic response is linearly related to concentration for all components over a 5-500 ng range. Analytical recovery of the drugs and metabolites from plasma is approximately 75 to 85% at low and high concentrations. Within-run and day-to-day precision (CV) is less than 5% for both high and low concentrations of most of these drugs and metabolites. Parallel analysis of clinical samples by gas chromatography indicates that results by the two techniques are comparable. We report some results of therapeutic monitoring of clinical samples.