bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain-specific proteins: solid-phase immunobioluminescence assay for neuron-specific enolase in human plasma.
Authors: Gerbitz K D , Summer J , Thallemer J
Published: January 2020

We raised specific antibodies in rabbits against pure alpha alpha- or gamma gamma-isoenzymes of enolase (EC 4.2.1.11) purified from human brain. After being specifically immunoabsorbed by the respective Sepharose-coupled enolase isoenzymes, the purified antibodies are desorbed by acidification and coated onto polystyrene tubes. Each sample containing the isoenzyme activity to be determined is incubated in the respective coated tube. The coating is then washed, and the reaction of the antibody-bound enzyme is initiated by adding 2-phosphoglycerate, ADP, and pyruvate kinase. The accumulation of ATP is measured by following the increase in light emission in the firefly luciferase bioluminescence system. The assay is as specific as the antibody used for coating. Its detection limit is about 5 X 10(-9) U, corresponding to about 0.1 pg or 10(-18) mol of enzyme protein per assay. Activities of enolase isoenzymes in human plasma can be evaluated separately, rapidly, and precisely. We used the assay to measure enolase isoenzyme activities in plasma of patients suffering from different types of malignant tumors.
Rapid semiquantitative measurement of total porphyrins in urine and feces by magnetic circular dichroism.
Authors: Ivanetich K M , Movsowitz C , Moore M R
Published: January 2020

Magnetic circular dichroism is a suitable technique for semiquantitative determination of total porphyrins in urine and feces, being rapid, reliable, and involving little sample preparation. The sensitivity of the assay suffices to distinguish normal from above-normal concentrations of total porphyrins in urine and feces. Results for urine compare well with those obtained by thin-layer chromatography of porphyrin esters (n = 42, r = 0.92) or ion-exchange column chromatography of porphyrin acids (n = 19, r = 0.80).
A highly sensitive affinity-column-mediated immunometric assay, as exemplified by digoxin.
Authors: Freytag J W , Dickinson J C , Tseng S Y
Published: January 2020

We describe a highly sensitive heterogeneous enzyme-linked immunoassay in which digoxin is used as the model analyte. An excess of enzyme-labeled monovalent antibody is incubated with sample containing the analyte such that all analyte is rapidly and quantitatively bound. Excess antibody that does not acquire a antigen in its binding site is rapidly removed from the mixture by passage through a porous affinity column containing immobilized analyte (or analog), present in vast excess. Only the labeled monovalent antibody that possesses an antigen in its binding site elutes from the column in the unbound fraction. The label present in this fraction is then quantified. Such an assay is extremely sensitive and obviates the limitations imposed by antibody affinity constants on homogeneous and competitive heterogeneous immunoassays. This assay can be performed rapidly and is readily amenable to automation.
Cell membrane abnormality detected in erythrocytes from patients with multiple sclerosis by partition in two-polymer aqueous-phase systems.
Authors: Van Alstine J M , Brooks D E
Published: January 2020

Erythrocytes from multiple sclerosis patients differ significantly (p less than 0.005) from those from controls with regard to hydrophobic affinity partition in two-polymer aqueous-phase systems containing dextran, poly(ethylene glycol) and poly (ethylene glycol)-fatty acid esters. The most likely source of the abnormality is the cell membrane.
Measurement of choriogonadotropin by chemiluminescence immunoassay and immunochemiluminometric assay: 1. Use of isoluminol derivatives.
Authors: Barnard G J , Kim J B , Brockelbank J L , Collins W P , Gaier B , Kohen F
Published: January 2020

We have developed immunoassays, monitored by the detection of chemiluminescence, for measuring choriogonadotropin in human urine. These methods involve the use of derivatives of isoluminol and include: (a) a labeled antigen with a second antibody covalently linked to polyacrylamide beads as the solid-phase reagent (i.e., solid-phase chemiluminescence immunoassay); and (b) an excess concentration of a specific antibody passively adsorbed onto the walls of polystyrene tubes and a labeled antibody of different specificity (i.e., two-site immunochemiluminometric assay). After the respective binding reactions, the solutions are aspirated, the antigen- or antibody-bound fractions are washed twice with 500 microL of buffer, sodium hydroxide (2 mol/L; 200 microL) is added, and the mixture is incubated for 60 min at 60 degrees C. After cooling, the label is oxidized with microperoxidase/hydrogen peroxide and the resulting chemiluminescence signal is measured for 10 s. We have evaluated the methods in terms of their sensitivity, precision, and clinical utility, and we compare results with values obtained by radioimmunoassay.
Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.
Authors: Lin H J , Wu P C , Lai C L , Chak W
Published: January 2020

A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.
Theoretical consideration of the effects of dilution on estimates of free thyroid hormones in serum.
Authors: Kamikubo K , Komaki T , Nakamura S , Sakata S , Yasuda K , Miura K
Published: January 2020

When concentrations of free thyroid hormones in serum are measured by equilibrium dialysis, the serum sample is diluted by dialysis buffer, resulting in a new equilibrium for distribution of these hormones that differs from that in the original serum sample. We have derived a mathematical formulation, based on an equilibrium model for multiple ligands and multiple binding sites, to determine the effect of dilution on the concentrations of free hormones in serum. Computer simulations based on the formulation indicate that both free thyroxin (T4) and free triiodothyronine (T3) are decreased by dilution of serum, free T3 being affected more than free T4. This dilution-related decrease is more prominent in hyperthyroidism than in euthyroidism, and even more striking for sera in which binding capacities for thyroid hormones are decreased (e.g., in thyroxin-binding globulin deficiency or hypoalbuminemia). These simulations can provide a basis for minimizing experimental errors in the estimates of concentrations of free thyroid hormones by equilibrium dialysis.
Glucocorticoids directly affect spectrophotometry of bilirubin in amniotic fluid.
Authors: Milwidsky A , Yagel S , Chaouat M , Mayer M
Published: January 2020

Dexamethasone or prednisolone, added in vitro to bilirubin-containing amniotic fluid, produces a time-dependent decrease in the 450-nm absorbance of the pigment. Neither the chemical determination of bilirubin in amniotic fluid nor the lecithin/sphingomyelin ratio as determined by thin-layer chromatography is affected by these glucocorticoids. The effect probably is not a result of displacement of bilirubin from its binding sites on albumin, because the absorbance of a solution of crystalline bilirubin at 450 nm is unaffected by added bovine serum albumin. Light scattering of amniotic fluid increases slightly when dexamethasone is added, whether or not low concentrations of bilirubin (less than 1.6 mumol/L) are present. Thus the effect on absorbance evidently is not ascribable to supersaturation and formation of a colloidal sol of bilirubin particles. This direct interference of glucocorticoids with the spectrophotometry of bilirubin in amniotic fluid prompts cautious interpretation of such data as an index to the severity of hemolytic disease of the fetus, specifically in cases of Rhesus-isoimmunization that are being treated with glucocorticoids.
Cytochrome b5 reductase activity in erythrocytes and leukocytes as related to sex and age.
Authors: Panin G , Pernechele M , Giurioli R , Secchieri S , Milanesi O , Pellegrino P A , Chiandetti L
Published: January 2020

We measured cytochrome b5 reductase (EC 1.6.2.2) activity in erythrocytes and leukocytes from subjects of both sexes ranging in age from neonate to adulthood, all either apparently healthy or without hematologic disorders. The activities were determined spectrophotometrically (J. Lab. Clin. Med. 72: 339-344, 1968). Within-run precision (CV) of the reductase determination was 6.4% and 3.7% for erythrocytes and leukocytes, respectively. Activity in either erythrocytes or leukocytes was unrelated to sex. Activity was lowest in erythrocytes of newborns (p less than 0.001) but reached adult values within the first postnatal year. Leukocyte activity was higher (p less than 0.05) in infants one to 24 months old than in other groups, possibly because of variations in leukocyte count in the first postnatal months.
Use and significance of reference serum as a secondary standard for electroimmunoassay of apolipoprotein A-I.
Authors: Fesmire J D , McConathy W J , Alaupovic P
Published: January 2020

This study was designed to show the effect of different primary standards and antisera on the variability of concentrations of apolipoprotein A-I (ApoA-I) in plasma and to document the usefulness of reference sera as a secondary standard in the electroimmunoassay of this apolipoprotein. We compared the reactivities of two separate ApoA-I samples and a preparation of high-density lipoprotein (HDL3) with seven different antisera to ApoA-I. The antisera reacted differently with each of the three standards, as shown by the different slopes of their standard curves. The resulting inconsistencies in the ApoA-I values for unknown plasma samples were shown to be corrected by the use of a reference serum. When ApoA-I analyses were carried out with the reference serum as a standard, all seven antisera gave statistically indistinguishable results for concentrations of plasma ApoA-I. The use of reference sera thus represents the simplest and most convenient means of standardizing the electroimmunoassay for ApoA-I.
Liquid-chromatographic determination of penbutolol and its principal metabolites in plasma and urine.
Authors: Miner D J , Binkley D A , Bechtol L D
Published: January 2020

We describe a sensitive, specific liquid-chromatographic determination of penbutolol and its 4-hydroxy metabolite in plasma and urine. The method involves a simple organic extraction, evaporation of the solvent, reconstitution in methanol/water, and injection into the chromatograph. Penbutolol, its metabolites, and the internal standard, propranolol, are resolved on a CN reversed-phase column and detected fluorometrically. Conjugates of penbutolol and its 4-hydroxy metabolite may be determined after a 2-h enzymic hydrolysis. Detection limits are in the range of 3 to 12 micrograms/L of plasma. The assay is reproducible and nearly free of interferences. Representative concentrations in blood and urine of normal volunteers are reported.
Semi-automated determination of the uremic toxin "b4-2".
Authors: Faguer P , Man N K , Pierrat D , Funck-Brentano J L
Published: January 2020

A semi-automated two-stage chromatographic system is described for use in evaluating neurotoxin b4-2 in body fluids of uremic patients and healthy subjects. The only manual step is the injection, thus decreasing the risk of operator error inherent in the previous manual method (Clin Chem 29: 703-707, 1983) and concurrently improving CV from 9% to 6.4% as measured during a 16-month period.
Atypical alkaline phosphatase isoenzyme in serum from a patient with Hodgkin's disease.
Authors: Beilby J P , Garcia-Webb P , Bhagat C I , Prins A
Published: January 2020

An alkaline phosphatase isoenzyme that did not move from the origin in agarose gel electrophoresis was detected in serum from a 51-year-old woman with Hodgkin's disease. Inhibitor and heat-inactivation studies of the patient's serum alkaline phosphatase showed properties resembling those of both liver and bone isoenzymes. No immunoglobulin or high-molecular-mass complexes with the alkaline phosphatase isoenzyme were detected. The relative molecular mass (Mr) of the atypical alkaline phosphatase isoenzyme was 182 000, that of the liver alkaline phosphatase isoenzyme control 170 000. Treatment of both of these isoenzymes with neuraminidase gave a product with an Mr of 140 000. We propose that a post-translational modification increased the carbohydrate content of the liver alkaline phosphatase isoenzyme, thus changing the charge characteristics of the enzyme and decreasing its electrophoretic mobility. We believe this to be the first report of a post-translational modification in a heat-sensitive isoenzyme of alkaline phosphatase.
High-density-lipoprotein cholesterol in heparin-MnCl2 supernates determined with the Dow enzymic method after precipitation of Mn2+ with HCO3-.
Authors: Bachorik P S , Walker R E , Virgil D G
Published: January 2020

Manganese interferes with enzymic cholesterol methods. In this study, we enzymically measured high-density-lipoprotein (HDL) cholesterol in heparin-Mn2+ supernates that had been treated with NaHCO3 (91 mmol/L) to precipitate Mn2+, and compared results with those by an automated Liebermann- Burchard method. For untreated supernates of 96 fresh plasma samples, the enzymic values were 10.4% higher than comparison-method values, a bias that declined to +2.3% for treated supernates. For 72 sera promptly frozen and stored after collection, the enzymic values for untreated and treated supernates were, respectively, 6.0% and 0.5% higher than comparison-method values. In all cases, the magnitude of the bias was independent of the concentrations of cholesterol, triglyceride, and HDL-cholesterol. Enzymic HDL-cholesterol measurements in NaHCO3-treated heparin-Mn2+ supernates prepared from four pooled serum controls agreed within 21 mg/L with values established for these pools by the Centers for Disease Control. We conclude that the accuracy of enzymic HDL cholesterol measurements in heparin-Mn2+ supernates in considerably increased by treatment with NaHCO3.
High-resolution densitometry: analysis of stained albumin bands as a model for electrophoresis of serum proteins.
Authors: Epstein D J , Neeley W E
Published: January 2020

In discussing the principles of quantitative analysis in thin-layer media, we show that requirements for quantitative analysis are not satisfied when stained protein electrophoretic bands are scanned with a conventional rectangular-slit densitometer. We investigated a high-resolution densitometer based on a linear photodiode array as an alternative analytical tool, using stained electrophoretic bands of radio-labeled human serum albumin as a simplified model for results of serum protein electrophoresis. Identical protein bands scanned with both the high-resolution densitometer and a conventional densitometer were quantified with improved accuracy and precision by the new instrument. We also used the high-resolution densitometer to develop a computer model for performance characteristics of a rectangular-slit densitometer.
Liquid-chromatographic measurement of amino acids in biological samples after formation of phenylthiohydantoin derivatives.
Authors: Biggs H G , Gentilcore L J
Published: January 2020

In this method for measuring amino acids in urine, serum, and tissue, the amino acids to be assayed and the internal standard, L-norleucine, are converted to phenylthiohydantoins , isolated by organic solvent extraction, separated by reversed-phase "high-pressure" liquid chromatography, and detected by ultraviolet absorbance. The analytes are identified by retention times and quantified by comparing peak heights with that of the internal standard. The peak-height ratios vary linearly with concentrations of 50 to 500 mg/L, corresponding to the concentration range usually found in biological samples. Detection limits are 5 to 20 mg/L. Inter- and intra-assay precision (CV) varies between 1 and 26%. Average analytical recoveries range between 67 and 100%.
Suitability of various control sera for use in testing the accuracy of cholesterol determination with enzymic kits.
Authors: Demacker P N , Boerma G J , Jansen A P
Published: January 2020

We used 20 different commercial enzymic kits to measure cholesterol in 19 commercial control sera and in a pooled specimen of human serum and compared the relative biases, with the Abell -Kendall procedure as reference. Our purpose was to select those sera in various enzymic kits showing behavior similar to that of human serum and which thus can be used to measure the accuracy of these kits. The overall mean relative biases obtained with each of the 20 kits for the pooled human serum on the one hand and for a given control serum on the other generally correlated significantly. On the basis of the correlation coefficients and regression equations, we could select the control sera best suited for measurement of accuracy. They were all human-serum based, with cholesterol concentrations greater than 5 mmol/L. Animal sera with above-normal lipid values produced by feeding special diets appeared to be less reliable control sera in this respect.
Evaluation of the EMIT reagent system for measurement of caffeine with the EMIT Lab 5000 System and a centrifugal analyzer.
Authors: Ou C N , Frawley V L , Ellis J M
Published: January 2020

Using a Syva EMIT Lab 5000 System, we evaluated a newly developed EMIT assay for measurement of caffeine in serum of neonates. The method is rapid, and coefficients of variation for within-run and day-to-day precision were less than 5%. The accuracy of the method, as evaluated from analytical recovery and a calibrator-crossover study, is excellent. Comparison of the results analyzed by the EMIT caffeine assay (y) and the liquid-chromatographic comparison method (x) yielded the linear regression equation y = 1. 052x + 0.293 (n = 110, r = 0.987, and Sy . x = 0.625). The method was also adapted to a COBAS-BIO centrifugal analyzer (y) and optimized to minimize sample and reagent consumption. Linear regression analysis yielded the equation y = 1. 031x + 0.227 (n = 61, r = 0.992, and Sy . x = 0.488). As many as 400 assays per EMIT kit can be performed with the analyzer method, with excellent precision and accuracy. The kit assay and this centrifugal analyzer are well suited for rapid batch testing: 28 analyses (including six calibrators) in 10 min.
Semiautomated analysis for mercury in whole blood, urine, and hair by on-stream generation of cold vapor.
Authors: Peter F , Strunc G
Published: January 2020

In this method for quantitative determination of mercury in blood, urine, and hair, the specimen is first digested in a mixture of solid potassium permanganate and concentrated sulfuric acid. Excess oxidizing agent is reduced by hydroxylamine hydrochloride. The mercury liberated with stannous chloride is quantified by measuring its absorbance at 254 nm. This method shortens digestion time considerably and ensures accurate and reproducible results. Reagents must be free of mercury contamination; every new lot must be checked before use. This system can reliably accommodate as many as 30 specimens per hour and is suitable for use in laboratories that analyze a large number of biological specimens for total mercury.
Free thyroxin index and direct measurements of free thyroxin compared for evaluating postpartum autoimmune thyroid dysfunction.
Authors: Jansson R , Forberg R , Levin K
Published: January 2020

Measurement of free thyroxin (FT4) by a recently introduced commercial assay (Amerlex Free T4 RIA) was compared with the calculated free thyroxin index (FT4I) for serum from 104 postpartum women. Of these, 63 had transient thyroid dysfunction due to autoimmune thyroiditis, six had transient Graves' thyrotoxicosis, and 35 were euthyroid with no signs of autoimmune thyroid disease. The correlation between results for FT4 and the calculated FTI for 95 serum samples from women with no signs of autoimmune thyroiditis (r = 0.941; p = 0.0001) was almost identical to that for 270 serum samples from women with thyroid microsomal autoantibodies characteristic of autoimmune thyroiditis (r = 0.937; p = 0.0001). Furthermore, we observed no difference when the autoimmune group was subdivided according to low or high titers of thyroid microsomal antibodies. In no case did autoantibodies to thyroxin interfere with the FT4 assay. However, one woman had a spuriously low value for FT4I owing to interference by autoantibodies to triiodothyronine with the triiodothyronine resin uptake test. We conclude that the FT4 RIA assay provided diagnostic information in this group of postpartum women equivalent to that of the more elaborate procedure of determining FT4I.
Simultaneous determination of penicillin and cephalosporin antibiotics in serum by gradient liquid chromatography.
Authors: Annesley T , Wilkerson K , Matz K , Giacherio D
Published: January 2020

We describe a "high-performance" liquid-chromatographic method for simultaneously measuring various penicillin and cephalosporin antibiotics. After extraction from serum, which in general is quantitative, the drugs are separated by use of a "Bondapak phenyl" column and a gradient mobile phase. For these drugs retention times depend on the pH of the mobile phase; we present retention times under selected pH conditions.
Enzymic assay for oxalate in unprocessed urine, as adapted for a centrifugal analyzer.
Authors: Urdal P
Published: January 2020

In this automated modification of the oxalate decarboxylase method, oxalate can be measured (12 per hour) in acidified but otherwise unprocessed urine. Standard curves are linear up to at least 2.5 mmol/L. When 0.50 mmol of oxalate was added per liter to samples of 18 patients' urines, a mean analytical recovery of 98.5% (SD 3.6%) was obtained. Within-series CVs were 3.4 and 1.0%, between-series CVs 7.3 and 2.7% (n = 15) for oxalate concentrations of 0.31 and 0.61 mmol/L. The lower limit of detection is 25 mumol/L. Concentrations measured with this "direct" method correlated well (r = 0.95) with those measured after precipitation with calcium and ethanol and resolubilization in dilute sulfuric acid. For 17 healthy volunteers the mean urinary excretion of oxalate was 0.37 (SD 0.14) mmol/24 h.
Assessment of analytical bias: four new ways to use recovery measurements.
Authors: Kemp G J
Published: January 2020

I describe three ways of using data from analytical-recovery studies to assess analytical nonlinearity, without access to samples of known concentration. I then describe a recovery-based method of assessing constant, proportional, and non-linear errors with use of as little as one sample pool of known concentration. In each case, I present the theoretical basis of the method and an outline of a practical experimental protocol.
Selenium concentrations in plasma of patients with arteriographically defined coronary atherosclerosis.
Authors: Moore J A , Noiva R , Wells I C
Published: January 2020

A prospective epidemiological study (Lancet ii: 175-179, 1982) implicates low concentrations of selenium in plasma in coronary atherogenesis. We examined this relationship more directly by fluorometry of selenium in the plasma of 91 hospitalized patients who were being examined by coronary arteriography for clinical evaluation of chest pain. We observed a significant, inverse correlation between the plasma selenium and severity of coronary atherosclerosis. These results confirm those of the epidemiological studies, but the role, if any, of selenium in atherogenesis still is unclear. Its concentration in plasma is decreased by ethanol and cigarette use; possibly this is the mechanism of its relation to hypertension and atherosclerosis.
Interlaboratory comparison of 25-hydroxyvitamin D determination.
Authors: Mayer E , Schmidt-Gayk H
Published: January 2020

This interlaboratory study on determination of 25-hydroxyvitamin D (25-OH-D) in serum involved 15 laboratories in eight European countries. All could distinguish between normal (50 +/- 31 nmol/L, mean +/- SD) and grossly increased concentrations, but for eight laboratories the results for serum samples with low and normal 25-OH-D content overlapped. In general, values were well reproducible, but interlaboratory variation in 25-OH-D measurement was large, 24,25(OH)2D3 interfering in most of the assays. We present evidence in favor of chromatography before assay, as opposed to nonchromatographic methods. Liquid chromatography with ultraviolet detection for quantifying 25-OH-D2 and 25-OH-D3 appears to be an appropriate reference method, whereas competitive protein binding assay is the method of choice for routine determinations. Control sera with subnormal, normal, and above-normal concentrations of 25-OH-D3 are needed for use in standardization of 25-OH-D assays.
A new endogenous immunoenzyme assay for prostatic acid phosphatase evaluated.
Authors: Babson A L
Published: January 2020

I evaluated the performance of the "ORTHO PAP-IA," an endogenous immunoenzyme assay for prostatic acid phosphatase. The procedure is based on double-antibody precipitation of the antigen, followed by quantification of its enzymic activity. The antibodies are in large excess, to speed the reactions and minimize sensitivity to variations in assay conditions. Enzymic activity is measured via an extremely sensitive colorimetric reaction, the analytical sensitivity of which exceeds that of radioimmunoassay. Absorbance is linearly related to activity concentration up to an absorbance of 4.0, and only a single calibration standard is required. Within-run CV was less than 2%, between-run CV about 4%. Neither individual blanks nor assay in duplicate is required. All reagents, including the enzyme standard, are stable solutions. Results correlated well with those by a standard radioimmunoassay (r = 0.993, n = 38).
Affinity chromatography used in distinguishing alpha-fetoprotein in serum from patients with tumors of hepatic parenchyma and of germ cells.
Authors: Buamah P K , Cornell C , Skillen A W
Published: January 2020

We used affinity chromatography on concanavalin A Sepharose to study the serum alpha-fetoprotein of 10 patients with histologically proven germ-cell tumors and 12 patients with primary liver cancer. Less than 50% of the fetoprotein from germ-cell tumors bound to concanavalin A, as compared with more than 80% of the alpha-fetoprotein from primary liver cancers.
Simultaneous determination of trace elements in serum by energy-dispersive x-ray fluorescence spectrometry.
Authors: Rastegar F , Maier E A , Heimburger R , Christophe C , Ruch C , Leroy M J
Published: January 2020

Energy-dispersive x-ray fluorescence is applied in the analysis of human serum to determine the concentrations of several elements simultaneously with minimal manipulation of the sample. The analytical procedure has been developed with standard sera, and standardization, detection limits, and reproducibility have been established. A 50-microL sample of diluted serum, to which an internal standard has been added, is deposited on a thin (4-microns thick) polypropylene film and analyzed by x-ray fluorescence. We report the statistical distributions of the concentrations of Fe, Cu, Zn, and Br obtained in the population (103 samples) studied, and report detection limits for the other 22 elements studied. The simplicity of the method, the high throughput, and the possibility of automating the measurements make this procedure suitable for screening large numbers of sera.
Carboxyhemoglobin, cotinine, and thiocyanate assay compared for distinguishing smokers from non-smokers.
Authors: Pojer R , Whitfield J B , Poulos V , Eckhard I F , Richmond R , Hensley W J
Published: January 2020

We compared cotinine, carboxyhemoglobin, and thiocyanate concentrations in blood sampled from 187 cigarette smokers and 181 non-smokers. All three differed significantly between smokers and non-smokers. Cotinine performed best as a test for assessing smoking status, with a sensitivity of 98% as compared with 94% for carboxyhemoglobin and 80% for thiocyanate, all at a specificity of 95%. These differences were statistically significant. Results by none of these three methods correlated well with number of cigarettes smoked per day.
An automated immunoradiometric assay for human thyrotropin.
Authors: John R , Jones M K
Published: January 2020

In this two-site immunoradiometric assay for thyrotropin, developed for use in the "Kemtek 3000" automated radioimmunoassay system, commercially available monoclonal antibody to thyrotropin is labeled with 125I, and the solid-phase antibody is an IgG fraction of sheep antiserum to thyrotropin, covalently coupled to reprecipitated aminocellulose. There are two incubations, totalling 3 h, the sensitivity is 0.03 milli-int. unit/L. The mean thyrotropin value for 82 healthy euthyroid subjects was 1.7 milli-int. units/L (range 0.4-3.6). For 19 overtly clinically and biochemically hyperthyroid subjects the values ranged from undetectable to 0.2 milli-int. unit/L. In this assay, euthyroid and hyperthyroid subjects can be distinguished with assay of a single basal sample. The assay appears suitable for routine use as a first-line test of thyroid function.
Highly sensitive immunoassays based on use of liposomes without complement.
Authors: Litchfield W J , Freytag J W , Adamich M
Published: January 2020

We describe a novel liposome-based immunoassay in which covalently linked hapten-cytolysin conjugates are used instead of complement and surface-immobilized immunoreagents. Stable, unilamellar liposomes containing entrapped alkaline phosphatase as a marker enzyme were prepared by dialysis of octyl glucoside from suspensions of cholesterol and egg yolk lecithin. The resulting vesicles could be immediately lysed by addition of either bee venom melittin or hapten-melittin conjugates. Using ouabain, an analog of digoxin, we synthesized conjugates that were more lytic than mellitin alone but that were inhibited in the presence of antibody. This inhibition was affected by adding competing free digoxin at various concentrations to obtain standard curves. The same liposome preparations could be lysed with a biotin-melittin conjugate, which was inhibited by avidin. The latter system was affected by free biotin and might be used to couple this approach to various heterogeneous immunoassays.
Detection of alpha 2-macroglobulin-associated proteases in the plasma of patients with rheumatoid arthritis.
Authors: Gaspar A , Skosey J L , Sequeira W , Teodorescu M
Published: January 2020

In previous work a polyclonal B cell activator has been detected in the serum of patients with rheumatoid arthritis (RA). This activator is associated with alpha 2-macroglobulin (alpha 2M) and its activity is blocked by low-Mr trypsin inhibitors, which suggests that it may be a protease-alpha 2M complex. Here we determined the possibility of developing a routine clinical chemistry test for detection of this complex in patients' blood. We measured with chromogenic substrates the total proteolytic activity of citrated plasma and of the alpha 2M immunoabsorbed from plasma. Low-Mr substrates containing Arg were degraded much better by plasma from RA patients than by plasma from patients with other arthritides. Low-Mr substrates containing Leu, Lys, or Gly or the large-Mr substrate Azocoll were not degraded by RA patients' plasma. alpha 2M from RA patients' plasma attached to a solid-phase immunoabsorbent degraded an Arg-containing tripeptide much better than did the alpha 2M from normal donors, from patients with systemic lupus erythematosus, or from those with joint inflammation of other, "non-autoimmune" origin. Although the enzyme associated with alpha 2M in the plasma from RA patients appeared to be similar to trypsin, the differences in optimal pH, cation concentration, degradation of Lys-containing substrates, and biological activity suggest otherwise. We speculate that the alpha 2M-protease complexes are generated in the immune system and contribute to the inflammatory and autoimmune phenomena in RA.
Quantification of salicylate in serum by use of salicylate hydroxylase.
Authors: You K , Bittikofer J A
Published: January 2020

In this enzymatic method for salicylate in serum, Pseudomonas salicylate hydroxylase (EC 1.14.13.1) is used. This stable enzyme catalyzes the stoichiometric, unidirectional conversion of salicylate and NAD(P)H to catechol and NAD(P)+ in the presence of molecular oxygen. The concentration of salicylate in a clinical sample is determined by measuring the delta A at 340 nm as compared with a standard. This new method is rapid, highly specific, requires 40 microL or less of sample, and we saw no interference by any of 61 commonly used drugs. Lipemic, icteric, or hemolyzed samples can be used. Furthermore, this method does not involve extraction, deproteinization, or derivatization. Results are precise and agree well with those obtained by the Trinder test.
Use of a rapid, sensitive immunoradiometric assay for thyrotropin to distinguish normal from hyperthyroid subjects.
Authors: Cobb W E , Lamberton R P , Jackson I M
Published: January 2020

Using a new three-site immunoradiometric assay for thyrotropin (TSH), we measured concentration of this hormone in the serum of 47 patients with hyperthyroidism and 46 controls. The mean and range of serum TSH concentration was significantly lower in thyrotoxic than in control subjects, and it was possible to correctly identify 96% of thyrotoxic patients on the basis of a serum TSH concentration less than 0.5 milli-int. unit/L. We conclude that such measurement is highly sensitive for distinguishing hyperthyroid from normal subjects, and that the lower limit of normal for TSH in serum is about 0.5 milli-int. unit/L.
Outcome Bias and Expertise in Investigations under International Humanitarian Law
Authors: Broude Tomer , Levy Inbar
Published: January 2020

Many international law decisions are made by individuals, often possessed with expertise, legal or otherwise. We examine individual international humanitarian law (IHL) decision-making on two levels: military decisions made ex ante regarding real-time operational questions under conditions of uncertainty and imperfect information, and subsequent ex post evaluations of the propriety of military decisions in the context of military investigations regarding legal responsibility with respect to proportionality and reasonableness. IHL requires ex post investigators to consider only information available at the time decisions were made. Through an experimental vignette study conducted with laypersons, legal experts and people with field experience, we test whether they are susceptible to cognitive ‘outcome bias’, specifically the extent to which the knowledge of operational outcomes, especially regarding incidental civilian harm, influences ex post normative evaluations. Our results demonstrate a general tendency towards outcome bias, which is somewhat tempered by expertise. Individuals with operational decision-making experience may be less prone to outcome bias than legal experts. We discuss possible implications for the design of military investigations relating to IHL.
Origin Recognition Complex (ORC) Evolution Is Influenced by Global Gene Duplication/Loss Patterns in Eukaryotic Genomes
Authors: Ocaña-Pallarès Eduard , Vergara Zaida , Desvoyes Bénédicte , Tejada-Jimenez Manuel , Romero-Jurado Ainoa , Galván Aurora , Fernández Emilio , Ruiz-Trillo Iñaki , Gutierrez Crisanto
Published: January 2020

The conservation of orthologs of most subunits of the origin recognition complex (ORC) has served to propose that the whole complex is common to all eukaryotes. However, various uncertainties have arisen concerning ORC subunit composition in a variety of lineages. Also, it is unclear whether the ancestral diversification of ORC in eukaryotes was accompanied by the neofunctionalization of some subunits, for example, role of ORC1 in centriole homeostasis. We have addressed these questions by reconstructing the distribution and evolutionary history of ORC1-5/CDC6 in a taxon-rich eukaryotic data set. First, we identified ORC subunits previously undetected in divergent lineages, which allowed us to propose a series of parsimonious scenarios for the origin of this multiprotein complex. Contrary to previous expectations, we found a global tendency in eukaryotes to increase or decrease the number of subunits as a consequence of genome duplications or streamlining, respectively. Interestingly, parasites show significantly lower number of subunits than free-living eukaryotes, especially those with the lowest genome size and gene content metrics. We also investigated the evolutionary origin of the ORC1 role in centriole homeostasis mediated by the PACT region in human cells. In particular, we tested the consequences of reducing ORC1 levels in the centriole-containing green alga Chlamydomonas reinhardtii. We found that the proportion of centrioles to flagella and nuclei was not dramatically affected. This, together with the PACT region not being significantly more conserved in centriole-bearing eukaryotes, supports the notion that this neofunctionalization of ORC1 would be a recent acquisition rather than an ancestral eukaryotic feature.
A hybrid Galerkin finite element method for seismic wave propagation in fractured media
Authors: Vamaraju Janaki , Sen Mrinal K , De Basabe Jonas , Wheeler Mary
Published: January 2020

The discontinuous Galerkin finite element method (DGM) is a promising algorithm for modelling wave propagation in fractured media. It allows for discontinuities in the displacement field to simulate fractures or faults in a model. Our approach is based on the interior-penalty formulation of DGM, and the fractures are simulated using the linear-slip model, which is incorporated into the weak formulation. On the other hand, the spectral element method (SEM) can be used to simulate elastic wave propagation in non-fractured media. SEM uses continuous basis functions which do not allow for discontinuities in the displacement field. However, the computation cost of DGM is significantly larger than SEM due primarily to increase in the number of degrees of freedom. Here we propose a hybrid Galerkin method (HGM) for elastic wave propagation in fractured media that combines the salient features of each of the algorithm resulting in significant reduction in computational cost compared to DGM. We use DGM in areas containing fractures and SEM in regions without fractures. The coupling between the domains at the interfaces is satisfied in the weak form through interface conditions. The degree of reduction in computation time depends primarily on the density of fractures in the medium. In this paper, we formulate and implement HGM for seismic wave propagation in fractured media. Using realistic 2-D/3-D numerical examples, we show that our proposed HGM outperforms DGM with reduced computation cost and memory requirement while maintaining the same level of accuracy.
Evaluating the most popular diabetes websites in the USA: a content analysis
Authors: Holtz Bree E
Published: January 2020

Over 1.5 million Americans are diagnosed with diabetes every year, and the majority of them go online to learn about their condition. Unfortunately, the online diabetes landscape is crowded, and the quality of website content is unknown—guidance for patients and healthcare providers regarding online diabetes information is needed. To address this, nine of the top diabetes websites in the USA were identified through a comprehensive, multiple-query search and a content analysis was conducted. Two coders, with high inter-coder reliability, evaluated these websites on six factors: (i) Design; (ii) Credibility; (iii) Accessibility; (iv) Literacy; (v) Engagement; and (vi) Diabetes Content. The analysis revealed that the websites presented most of the assessed design features. Few of the sites scored well on the credibility assessment, while the majority of sites did not note an author, editor or reviewer. With regard to accessibility, less than half of the items we assessed were met. Furthermore, the websites possessed wide variability in literacy, with an average readability grade level of 8.8. Most of the websites fared well in engagement, demonstrating multiple avenues to interact with the organization. All of the websites provided basic information regarding diabetes, but many did not include information for caregivers, providers or insurance information. In conclusion, patients and their family members will continue to seek online information about diabetes, its effects and treatment options. Therefore, this multiple-metric evaluation provides essential information for these individuals, and healthcare providers, who can now give better informed website recommendations to their patients and families.
Genomic dissection of 43 serum urate-associated loci provides multiple insights into molecular mechanisms of urate control
Authors: Boocock James , Leask Megan , Okada Yukinori ,   Asian Genetic Epidemiology Network (AGEN) Consortium, Matsuo Hirotaka , Kawamura Yusuke , Shi Yongyong , Li Changgui , Mount David B , Mandal Asim K , Wang Weiqing , Cadzow Murray , Gosling Anna L , Major Tanya J , Horsfield Julia A , Choi Hyon K , Fadason Tayaza , O’Sullivan Justin , Stahl Eli A , Merriman Tony R
Published: January 2020

High serum urate is a prerequisite for gout and associated with metabolic disease. Genome-wide association studies (GWAS) have reported dozens of loci associated with serum urate control; however, there has been little progress in understanding the molecular basis of the associated loci. Here, we employed trans-ancestral meta-analysis using data from European and East Asian populations to identify 10 new loci for serum urate levels. Genome-wide colocalization with cis-expression quantitative trait loci (eQTL) identified a further five new candidate loci. By cis- and trans-eQTL colocalization analysis, we identified 34 and 20 genes, respectively, where the causal eQTL variant has a high likelihood that it is shared with the serum urate-associated locus. One new locus identified was SLC22A9 that encodes organic anion transporter 7 (OAT7). We demonstrate that OAT7 is a very weak urate-butyrate exchanger. Newly implicated genes identified in the eQTL analysis include those encoding proteins that make up the dystrophin complex, a scaffold for signaling proteins and transporters at the cell membrane; MLXIP that, with the previously identified MLXIPL, is a transcription factor that may regulate serum urate via the pentose–phosphate pathway and MRPS7 and IDH2 that encode proteins necessary for mitochondrial function. Functional fine mapping identified six loci (RREB1, INHBC, HLF, UBE2Q2, SFMBT1 and HNF4G) with colocalized eQTL containing putative causal SNPs. This systematic analysis of serum urate GWAS loci identified candidate causal genes at 24 loci and a network of previously unidentified genes likely involved in control of serum urate levels, further illuminating the molecular mechanisms of urate control.
DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism
Authors: Maresca Alessandra , Del Dotto Valentina , Capristo Mariantonietta , Scimonelli Emanuela , Tagliavini Francesca , Morandi Luca , Tropeano Concetta Valentina , Caporali Leonardo , Mohamed Susan , Roberti Marina , Scandiffio Letizia , Zaffagnini Mirko , Rossi Jacopo , Cappelletti Martina , Musiani Francesco , Contin Manuela , Riva Roberto , Liguori Rocco , Pizza Fabio , La Morgia Chiara , Antelmi Elena , Loguercio Polosa Paola , Mignot Emmanuel , Zanna Claudia , Plazzi Giuseppe , Carelli Valerio
Published: January 2020

ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders, and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement, we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients. We documented impaired activity of purified DNMT1 mutant proteins, which in fibroblasts results in increased DNMT1 amount. We demonstrated that DNMT1 is not localized within mitochondria, but it is associated with the mitochondrial outer membrane. Concordantly, mitochondrial DNA failed to show meaningful CpG methylation. Strikingly, we found activated mitobiogenesis and OXPHOS with significant increase of H2O2, sharply contrasting with a reduced ATP content. Metabolomics profiling of mutant cells highlighted purine, arginine/urea cycle and glutamate metabolisms as the most consistently altered pathways, similar to primary mitochondrial diseases. The most severe mutations showed activation of energy shortage AMPK-dependent sensing, leading to mTORC1 inhibition. We propose that DNMT1 RFTS mutations deregulate metabolism lowering ATP levels, as a result of increased purine catabolism and urea cycle pathways. This is associated with a paradoxical mitochondrial hyper-function and increased oxidative stress, possibly resulting in neurodegeneration in non-dividing cells.
Stable oxygen isotope reconstruction of temperature exposure of the Icelandic cod (Gadus morhua) stock over the last 100 years
Authors: von Leesen Gotje , Ninnemann Ulysses S , Campana Steven E
Published: January 2020

Increasing water temperatures are predicted around the globe, with high amplitudes of warming in Subarctic and Arctic regions where Atlantic cod (Gadus morhua) populations currently flourish. We reconstructed oxygen isotope and temperature chronologies from Icelandic cod otoliths, one of the largest cod stocks in the world, to determine if cod moved or migrated over the last 100 years to avoid increasing water temperatures. For δ18Ootolith analysis, individual annual growth increments from immature and mature life history stages were micromilled from adult otoliths, which were collected in southern Iceland. Linear mixed-effect models confirmed that stable oxygen isotope time series of immature and mature cod differ significantly between both life stages (p < 0.001). Overall, cod otolith δ18O was significantly correlated with water temperature (sea surface temperature: p < 0.001, temperature at 200 m depth: p < 0.01), indicating that Atlantic cod were exposed to fluctuating water temperatures during the past 100 years and did not move as a response to increasing ocean temperatures. All of the alternate physical factors that were considered for the isotope-based variation in the temperature exposure of Icelandic cod were rejected.
Late cardiovascular complication after varicose vein endovascular ablation
Authors: Fiore Antonio , Piscitelli Mariantonietta , Radu Costin , Folliguet Thierry
Published: January 2020

Venous insufficiency of the lower limbs is one of the most common vascular disorders affecting millions of people worldwide. Endovascular techniques are considered by current guidelines as simple, safe and effective. Persistence or even migration of foreign bodies after varicose vein endovascular surgery is a rare and unfeared complication. Herein, we present the case of a 39-year-old woman who underwent endoluminal treatment of varicose veins >2 years ago and she was admitted to our department for late cardiovascular complication caused by catheter or guidewire rupture and the dissemination of its fragments in the inferior vena cava, right heart chambers, liver and pulmonary arteries. A systematic verification of the length and integrity of the devices should be performed at the end of every endovascular procedure.