bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonlinear optical absorption and asymmetric charge carrier conduction in chemical vapor deposited single-layer graphene
Authors: Thomas Subin , Nalini Savitha , Jayaraj M K , L S Vikas , K Rajeev Kumar
Published: January 2020

In this work, we report the nonlinear optical absorption and asymmetric charge carrier conduction in single layer graphene films deposited by chemical vapor deposition (CVD) technique on copper foils with pretreated surface. XRD texture and pole figure analysis of the substrate are utilized for the visualization of the effect of the pretreatment on the substrate. The synthesised graphene is employed as a channel layer in a back gated field-effect transistor and the asymmetric behavior of charge carriers is analyzed. Nonlinear optical response of graphene is recorded after transferring it onto a quartz substrate. Open aperture Z-scan technique yields a nonlinear absorption coefficient of 5.34 × 106 cm GW−1. The film exhibits saturable absorption in the visible range with a saturation intensity as low as 0.134 GW cm−2.
Mechanism of silver/glass interaction in the metallization of crystalline silicon solar cells
Authors: Sun Xinjie , Yao Shanshan , Xing Juanjuan , Zhang Jiefeng , Yang Yunxia , Li Hongbo , Tong Hua , Yuan Xiao
Published: January 2020

In order to reveal the interaction between solid substances (Ag powder and glass frit) during the metallization of crystalline silicon solar cells, the effect of glass frit on the Ag powder sintering and the mechanism of Ag colloid formation in glass phase were investigated. The results show that the Ag powder could grow into larger grains as mixed with a certain amount of glass frit, because the corrosion of Ag by glass melt enabled early start of Ag sintering. This makes the internal grain size and electric resistivity of sintered Ag lines to be closely related to the glass content in the silver paste. Subsequently, it was found that the formation of Ag colloid in glass phase is a physical process rather than a chemical transformation process. Taking Pb-B-Zn-Al composite oxide glasses for exemplification, a feasible strategy of raising the solubility of Ag in glass melt is proposed.
Bilateral Breast Reconstruction With Profunda Artery Perforator Flaps and Immediate Implant Augmentation
Authors: Chu Carrie K , DeFazio Michael , Largo Rene D , Ross Merrick
Published: January 2020

The smaller volume of the profunda artery perforator (PAP) flap relative to that of abdominal flaps limits the size of breast reconstruction that may be achieved. Immediate implant augmentation of abdominal free flaps has been performed, but immediate implant augmentation of PAP flaps has never been described. A 54-year-old woman with BRCA2 mutation, submuscular implants, and previous abdominoplasty presented for nipple-sparing mastectomies (NSM). Autologous tissue volume was inadequate to support reconstruction to the desired size. She wished to avoid serial expansion. Skin quality was unsuitable for direct-to-implant reconstruction. The patient underwent bilateral NSM. The previous implants were removed with capsule preservation. Bilateral PAP flaps were harvested and anastomosed to the internal mammary vessels. Moderate classic profile 170-mL smooth round silicone implants were placed into the existing capsule pockets with lateral capsulorraphy. There were no flap, implant, or infectious complications. Initial mastectomy skin and nipple ischemia completely resolved without necrosis. Donor site healing was uneventful. At 8 months, the reconstruction is supple and the implants remain well-positioned without rippling. One minor revision was performed for fat grafting and to correct lateral nipple deviation. PAP flap breast reconstruction with immediate implant augmentation is technically feasible. Advantages include improved prosthetic coverage, allowing for immediate reconstruction to a larger size with reduced concern regarding mastectomy skin necrosis and threat to the device, optimal implant camouflage, and improved substrate for secondary fat grafting if necessary.

Level of Evidence: 5
Bridging-induced microphase separation: photobleaching experiments, chromatin domains and the need for active reactions
Authors: Brackley C A , Marenduzzo D
Published: January 2020

We review the mechanism and consequences of the ‘bridging-induced attraction’, a generic biophysical principle that underpins some existing models for chromosome organization in 3D. This attraction, which was revealed in polymer physics-inspired computer simulations, is a generic clustering tendency arising in multivalent chromatin-binding proteins, and it provides an explanation for the biogenesis of nuclear bodies and transcription factories via microphase separation. Including post-translational modification reactions involving these multivalent proteins can account for the fast dynamics of the ensuing clusters, as is observed via microscopy and photobleaching experiments. The clusters found in simulations also give rise to chromatin domains that conform well with the observation of A/B compartments in HiC experiments.
incaRNAfbinv 2.0: a webserver and software with motif control for fragment-based design of RNAs
Authors: Retwitzer Matan Drory , Reinharz Vladimir , Churkin Alexander , Ponty Yann , Waldispühl Jérôme , Barash Danny
Published: January 2020

RNA design has conceptually evolved from the inverse RNA folding problem. In the classical inverse RNA problem, the user inputs an RNA secondary structure and receives an output RNA sequence that folds into it. Although modern RNA design methods are based on the same principle, a finer control over the resulting sequences is sought. As an important example, a substantial number of non-coding RNA families show high preservation in specific regions, while being more flexible in others and this information should be utilized in the design. By using the additional information, RNA design tools can help solve problems of practical interest in the growing fields of synthetic biology and nanotechnology. incaRNAfbinv 2.0 utilizes a fragment-based approach, enabling a control of specific RNA secondary structure motifs. The new version allows significantly more control over the general RNA shape, and also allows to express specific restrictions over each motif separately, in addition to other advanced features.

Availability and implementation

incaRNAfbinv 2.0 is available through a standalone package and a web-server at https://www.cs.bgu.ac.il/incaRNAfbinv. Source code, command-line and GUI wrappers can be found at https://github.com/matandro/RNAsfbinv.

Supplementary information

Supplementary data are available at Bioinformatics online.
Lineage EM algorithm for inferring latent states from cellular lineage trees
Authors: Nakashima So , Sughiyama Yuki , Kobayashi Tetsuya J
Published: January 2020

Phenotypic variability in a population of cells can work as the bet-hedging of the cells under an unpredictably changing environment, the typical example of which is the bacterial persistence. To understand the strategy to control such phenomena, it is indispensable to identify the phenotype of each cell and its inheritance. Although recent advancements in microfluidic technology offer us useful lineage data, they are insufficient to directly identify the phenotypes of the cells. An alternative approach is to infer the phenotype from the lineage data by latent-variable estimation. To this end, however, we must resolve the bias problem in the inference from lineage called survivorship bias. In this work, we clarify how the survivorship bias distorts statistical estimations. We then propose a latent-variable estimation algorithm without the survivorship bias from lineage trees based on an expectation–maximization (EM) algorithm, which we call lineage EM algorithm (LEM). LEM provides a statistical method to identify the traits of the cells applicable to various kinds of lineage data.

Availability and implementation

An implementation of LEM is available at https://github.com/so-nakashima/Lineage-EM-algorithm.

Supplementary information

Supplementary data are available at Bioinformatics online.
Generalizable sgRNA design for improved CRISPR/Cas9 editing efficiency
Authors: Hiranniramol Kasidet , Chen Yuhao , Liu Weijun , Wang Xiaowei
Published: January 2020

Motivation

The development of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has provided a simple yet powerful system for targeted genome editing. In recent years, this system has been widely used for various gene editing applications. The CRISPR editing efficacy is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage. While there have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA potency and generalizability across various experimental conditions.

Results

We employed a unique plasmid library expressed in human cells to quantify the potency of thousands of CRISPR/Cas9 sgRNAs. Differential sequence and structural features among the most and least potent sgRNAs were then used to train a machine learning algorithm for assay design. Comparative analysis indicates that our new algorithm outperforms existing CRISPR/Cas9 sgRNA design tools.

Availability and implementation

The new sgRNA design tool is freely accessible as a web application, http://crispr.wustl.edu.

Supplementary information

Supplementary data are available at Bioinformatics online.
Autophagy-mediated regulation of neutrophils and clinical applications
Authors: Yu Yao , Sun Bingwei
Published: January 2020

Autophagy, an adaptive catabolic process, plays a cytoprotective role in enabling cellular homeostasis in the innate and adaptive immune systems. Neutrophils, the most abundant immune cells in circulation, are professional killers that orchestrate a series of events during acute inflammation. The recent literature indicates that autophagy has important roles in regulating neutrophil functions, including differentiation, degranulation, metabolism and neutrophil extracellular trap formation, that dictate neutrophil fate. It is also becoming increasingly clear that autophagy regulation is critical for neutrophils to exert their immunological activity. However, evidence regarding the systematic communication between neutrophils and autophagy is insufficient. Here, we provide an updated overview of the function of autophagy as a regulator of neutrophils and discuss its clinical relevance to provide novel insight into potentially relevant treatment strategies.
The Effect of Pregnancy on the Pharmacokinetics of Total and Unbound Dolutegravir and Its Main Metabolite in Women Living With Human Immunodeficiency Virus
Authors: Bollen Pauline , Freriksen Jolien , Konopnicki Deborah , Weizsäcker Katharina , Hidalgo Tenorio Carmen , Moltó José , Taylor Graham , Alba-Alejandre Irene , van Crevel Reinout , Colbers Angela , Burger David ,   Pharmacokinetics of ANtiretroviral agents in HIV-infected pregNAnt women Network, Nellen J , Lyons F , Lambert J , Wyen C , Faetkenheuer G , Rockstroh J K , Schwarze-Zander C , Tariq Sadiq S , Gilleece Y , Wood C , Schalkwijk S , Bukkems V
Published: January 2020

Background

Pharmacokinetic and efficacy data on dolutegravir in pregnant women living with human immunodeficiency virus (HIV) are still limited but needed to support its use as one of the preferred antiretroviral agents.

Methods

Within the multicenter Pharmacokinetics of ANtiretroviral agents in HIV-infected pregNAnt women (PANNA) study, pregnant women living with HIV and using dolutegravir once daily (50 mg, with food) underwent 24-hour pharmacokinetic profiling in their third trimester and postpartum. Dolutegravir exposure in the third trimester was considered adequate if geometric mean unbound, pharmacologically active, minimal plasma concentrations (Cmin, unbound) and ≥90% of individual Cmin, unbound levels were >0.85 µg/L, the proposed 90% inhibitory concentration for unbound dolutegravir. Geometric mean ratios (GMRs) with 90% confidence intervals (CIs) for comparison of total and unbound pharmacokinetic parameters in the third trimester and postpartum were calculated, including the metabolic ratio for dolutegravir-glucuronide. Safety and virological data were collected.

Results

Seventeen women (76% black) were enrolled (25 evaluable pharmacokinetic profiles; 15 in the third trimester, 10 in postpartum). In the third trimester, geometric mean (coefficient of variation, %) Cmin, unbound was 2.87 (87) µg/L and 93% of individual Cmin, unbound levels were >0.85 µg/L. The GMR (90% CI) in the third trimester vs postpartum was 0.86 (.68–1.10) for area under the curve (AUC0-24h), and for Cmax, 0.93 (.77–1.13). GMR (90% CI) for the trough concentrations was 0.71 (.49–1.02), based on total dolutegravir concentrations. Four serious adverse events were reported, unlikely related to dolutegravir. The HIV polymerase chain reaction test was negative in 14/17 infants (result unknown for 3 infants).

Conclusions

Pharmacokinetic changes for dolutegravir in late pregnancy are not clinically relevant and support the use of dolutegravir 50 mg once daily with food in pregnancy.

Clinical Trials Registration

NCT00825929.
Alternative statistical approach to evaluating interlaboratory performance.
Authors: Ehrmeyer S S , Laessig R H
Published: January 2020

We report a new technique for realistic assessment of laboratory performance as measured by proficiency testing. Interlaboratory results accumulated from 129 participants during 18 months provide the baseline data from which we established "state-of-the-art" performance criteria for three ranges of pH, pCO2, and pO2. By concurrent use of two statistical measurement techniques, the cumulative percentile rank and the algebraic and absolute mean error, laboratories can accurately evaluate their performance in terms of acceptable state-of-the-art criteria, total error, or medical usefulness. The approach facilitates assessment of the nature of the errors that have led to inferior performance and identification of probable areas where improvement is possible. If criteria based on regulatory standards or medical usefulness goals are included, the system can provide a basis for licensure or professional quality improvement.
Pitfalls in, and new methods for, the radioimmunoassay for digoxin in urine.
Authors: O'Brien M S , Huston J R , Reid R J , Gibson T P
Published: January 2020

Examining techniques for radioimmunoassay of digoxin in urine, we found that the apparent concentration of digoxin is affected by endogenous substances, independent of the concentration of digoxin itself. We describe a modification of the Becton Dickinson solid-phase RIA that makes it more nearly accurate and more easily performed, and obviates the endogenous interference. Digoxin concentrations from 5.0 to at least 1000 micrograms/L can be measured.
Determination of plasma beta-endorphin and beta-lipotropin by cation-exchange liquid chromatography and radioimmunoassay.
Authors: Laatikainen T , Salminen K , Stenman U H , Leppäluoto J
Published: January 2020

In this method for rapid separation of beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) in plasma, most of the plasma proteins in a 0.5- to 2.0-mL sample of plasma are precipitated with acetonitrile at pH 4.7. beta-EP and beta-LPH in the supernate are completely separated by liquid chromatography on a cation-exchange column, with gradient elution with volatile buffers, and are eluted in 1.5- to 2.0-mL volumes. After lyophilization, the redissolved beta-EP and beta-LPH are determined by radioimmunoassay. The respective mean concentrations of immunoreactive beta-EP and beta-LPH in plasma of nonpregnant women were 4.0 (SD 2.0) and 5.1 (SD 3.0) pmol/L; during labor, 33.3 (SD 25) and 69.6 (SD 41) pmol/L; and in venous plasma from umbilical cords after spontaneous labor, 38.6 (SD 18) and 38.7 (SD 19) pmol/L.
Radioimmunoassay for 1,25-dihydroxyvitamin D in serum or plasma.
Authors: De Leenheer A P , Bauwens R M
Published: January 2020

For this sensitive RIA for 1 alpha,25-dihydroxyvitamin D, we used antibodies to 1 alpha,25-dihydroxycholecalciferol-3-hemisuccinate conjugated to bovine serum albumin, raised in rabbits. These antibodies show a high affinity for 1 alpha,25-dihydroxyvitamin D3 but cross react with other vitamin D metabolites as well. Extraction and liquid chromatography are required to isolate the 1 alpha,25-dihydroxyvitamin D from human serum or plasma with benzene (analytical recovery, 96%) and chromatographed by "high-performance" liquid chromatography on a silica column. The sensitivity of the assay is 2 pg/tube. The between-assay CV is 12.6% (means = 61.8 ng/L, n = 27) and the within-assay CV is 7.4% (means = 61.8 ng/L, n = 15). The reference interval, obtained from data on serum of healthy persons, is 51.8 +/- 15.1 ng/L (means +/- SD, n = 91), with no sex-related difference. Patients with chronic renal failure have lower concentrations in serum, and patients suffering from calcium nephrolithiasis have increased concentrations. Our assay offers the advantage of a relatively short prepurification procedure and the production of large amounts of stable gamma-globulins that can be used for several years without loss of binding properties.
A compound from uremic plasma and from normal urine isolated by liquid chromatography and identified by nuclear magnetic resonance.
Authors: Gallice P , Monti J P , Crevat A , Durand C , Murisasco A
Published: January 2020

A compound present in normal urine and in ultrafiltrates of uremic plasma in the fraction of so-called "uremic middle molecules" was isolated by liquid chromatography. Preliminary studies, including amino acid analysis, characterization of uronic acids, and ultraviolet spectroscopy, show that the molecule contains glycine, a uronic acid, and an aromatic ring. Characterization by 1H and 13C nuclear magnetic resonance spectrometry shows conclusively that this compound is a double conjugate of glucuronidate--o-hydroxyhippuric acid, which has been previously described by Zimmerman et al., using quite different techniques of isolation and identification (Clin Nephrol 14: 107, 1980; FEBS Lett 129: 237, 1981).
Kinetic determination of blood coagulation Factor XIII in plasma.
Authors: Muszbek L , Polgár J , Fésüs L
Published: January 2020

We have designed a new kinetic assay for estimating Factor XIII in plasma. Plasma fibrinogen is removed by treatment with bentonite (colloidal aluminum silicate) before measurement. During the lag phase, Factor XIII is transformed by thrombin and Ca2+ into active transglutaminase (EC 2.3.2.13), which attaches the substrate ethylamine to a glutamine residue in acetylated, dephosphorylated beta-casein. During the reaction, ammonia is released, which can be continuously monitored in an NADPH-dependent indicator reaction catalyzed by glutamate dehydrogenase (EC 1.4.1.4). We determined the optimal concentrations of substrate and activator and found that, to eliminate the clottable fibrinogen from the plasma samples, bentonite treatment was more advantageous than the traditional heat treatment. Results by the method correlate well with those by the most widely used amine incorporation and immunoinhibition assays for Factor XIII. We established a reference interval of 12.1-22.7 U/L; at optimal conditions, the variance of the method was less than 3% within this range. The method has several theoretical and practical advantages over traditional determinations of Factor XIII.
Uremic toxins, and their effect on intermediary metabolism.
Authors: Wills M R
Published: January 2020

In the late stages of chronic renal damage the functional mass of the kidney is reduced and there is progression to renal insufficiency, usually called uremia, in which all aspects of renal function are affected. The complexity of the biochemical aspects of the syndrome of uremia is a manifestation of the wide variety and nature of the individual disorders that contribute to the pathogenesis of the final clinical syndrome. One major feature is the retention of metabolic end products and their effects, as toxins, on intermediary metabolism. The retained end products, working singly or in combination, probably affect metabolic pathways by some modification of enzymic reactions. They act at the cell membrane level. Although "middle molecules" have been incriminated as uremic toxins, recent attention has also focused on trace elements--especially aluminum, which has been implicated in the pathogenesis of two major disorders, osteomalacic dialysis osteodystrophy and dialysis encephalopathy.
Indican interference with six commercial procedures for measuring total bilirubin.
Authors: Poon R , Hinberg I H
Published: January 2020

We have studied the effect of indican on six commercial procedures for the measurement of total bilirubin in serum. Total bilirubin measured by the Bilirubin A-Gent (Abbott) 2,4-dichlorophenyl diazonium procedure increased by 50 mg/L for each 1 mmol/L of added indican. Similarly, total bilirubin measured by the Bilirubin C-System (Boehringer Mannheim) 2,5-dichlorophenyl diazonium procedure increased by 33 mg/L per mmol/L of indican. Indican also interfered with the Micro Bilirubin Reagent Set (Harleco) Malloy-Evelyn procedure, but to a much lesser extent. The Jendrassik Bilirubin Reagent System (American Monitor) and a modified Jendrassik-Grof procedure (Hoffmann-LaRoche) adapted to the Cobas Bio analyzer were unaffected by the presence of indican. The amount of interference with the 2,5-dichlorophenyl diazonium procedure increased significantly with color development time and was twice the initial amount after 30 min. Concentrations of indican as high as 0.38 mmol/L have been found in sera of patients with renal failure, which would increase total bilirubin values measured by the first two procedures above by 19 and 12 mg/L, respectively. Users of these procedures should therefore be suspicious of unexpectedly high bilirubin values obtained with sera from patients with chronic renal disease.
Thromboplastic activity of cerebrospinal fluid in neurological disease.
Authors: Tutuarima J A , Hische E A , van Trotsenburg L , van der Helm H J
Published: January 2020

Estimates of thromboplastic activity in 1100 samples of cerebrospinal fluid indicate that an increased activity of this clotting factor is a nonspecific indicator of abnormality in the central nervous system, much like (e.g.) an increased count of mononuclear cells and an increased protein content. However, the proportion of abnormal results obtained by these three tests can differ markedly in different neurological disorders. Increased thromboplastic activity is about 14-fold more common in bacterial meningitis than in viral meningitis; thus the thromboplastin determination can be of value in discriminating between bacterial and viral meningitis.
Peroxidase-coupled method for kinetic colorimetry of total creatine kinase activity in serum.
Authors: Wimmer M C , Artiss J D , Zak B
Published: January 2020

We describe a peroxidase-coupled method involving a colorimetric indicator reaction for determining the total activity of creatine kinase (EC 2.7.3.2) in serum. The kinetically favorable reverse reaction is exploited to generate adenosine 5'-triphosphate, which is used in the glycerol kinase-catalyzed phosphorylation of glycerol. The glycerol 3-phosphate so generated is oxidized in the presence of alpha-glycerophosphate oxidase to produce hydrogen peroxide, which is reduced in the presence of peroxidase with the simultaneous oxidation and coupling of 4-aminoantipyrene and 2-hydroxy-3,5-dichlorobenzenesulfonate to produce an intensely colored red chromogen. Results of the proposed method (y) correlate well with those of the Boehringer-Mannheim "CK-NAC UV" method as applied to the Hitachi 705 chemistry analyzer (y = 1.025 chi - 18.1, r = 0.9985, n = 100, range = 19-4531 U/L). The sensitivity of the method, based on molar absorptivities, is nearly fourfold that of procedures involving the reduction of NADP+.
Comprehensive study of a thyroxin-analog-based assay for free thyroxin ("Amerlex FT4").
Authors: Wilkins T A , Midgley J E , Barron N
Published: January 2020

The basic theory of thyroxin-analog-based radioimmunoassays for free thyroxin has been extended to evaluate definitively the effects arising from residual binding of the tracer analog to serum proteins. Using experimentally determined binding constants and computer simulation techniques, we studied the effects of thyroxin-analog binding to serum proteins on results of Amerlex FT4 radioimmunoassay, using this improved mathematical model. Results from computer-simulation studies were compared both directly with in vitro experimental results and indirectly with clinical studies. Agreement was good among all three approaches. The relatively weak binding of analog to thyroxin-binding globulin and prealbumin does not significantly perturb Amerlex FT4 assay results. Binding of the analog by albumin has a small but quantifiable effect on assay results, amounting to an intrinsic bias of 0.08 pmol of free thyroxin per liter per gram of albumin per liter for euthyroid serum samples. This bias is unlikely to be important for most clinical laboratory samples, but it may be significant when one is interpreting results for those rare patients with genetic albumin abnormalities such as analbuminemia or familial dysalbuminemic hyperthyroxinemia. Massively increased concentrations of nonesterified fatty acids (e.g., after treatment with heparin) will lead to a spurious increase in free thyroxin in this and most other techniques, including equilibrium dialysis.
Monoclonal antibodies can precipitate low-density lipoprotein. I. Characterization and use in determining apolipoprotein B.
Authors: Marcovina S , France D , Phillips R A , Mao S J
Published: January 2020

We produced 20 mouse monoclonal antibodies against human plasma low-density lipoprotein (LDL). Individually they failed to precipitate LDL in agarose gel by the double-immunodiffusion technique; collectively they did, or as few as two combined monoclonal antibodies could do so. To mimic polyclonal antibodies in determination of apolipoprotein B (apo B) by radial immunodiffusion, a combination of four particular monoclonal antibodies (clones A, B, C, and D) was necessary. We characterized these four clones with respect to temperature dependency, affinity, total binding to 125I-labeled LDL, and specificity to the different species of apolipoprotein B. Two monoclonal antibodies (B and C) bound 100% of 125I-labeled LDL; clones A and D bound 80% and 87%, respectively. All four clones bound maximally to LDL at 4 degrees C. The affinity constants for clones A, B, C, and D were 0.6, 2.1, 3.8, and 2.3 X 10(9) L/mol, respectively. By the Western blotting technique, the four monoclonal antibodies all reacted with the species B-100 and B-74 of apolipoprotein B, and to various degrees with B-48 and B-26. Radial immunodiffusion (chi) and direct enzyme-linked immunosorbent assay (y) with a mixture of the four monoclonal antibodies gave almost identical results for 70 patients: y = 0.921 chi-2.58; r = 0.933.
Liquid chromatography of free hydroxyproline in serum.
Authors: Turpeinen U , Pomoell U M
Published: January 2020

We report a "high-performance" liquid-chromatographic method for measuring free hydroxyproline in serum. After separating the free hydroxyproline from the protein- and peptide-bound forms by centrifugal ultrafiltration, we derivatize the free form with 4-dimethylaminoazobenzene-4'-sulfonyl ("dabsyl") chloride and separate and quantify the derivative on a C18 chromatographic column. The mobile phase is sodium acetate buffer and acetonitrile, in a multilinear solvent gradient. The derivative is detected at 436 nm. Mean analytical recovery was 73%. The assay curve is linear from 14 to 213 pmol of hydroxyproline per 5-microL injection. The within- and interassay CVs were 8% at 10.7 mumol/L hydroxyproline and 10% at 13.5 mumol/L hydroxyproline, respectively.
Liquid-chromatographic assay of diazepam and its major metabolites in serum, and application to pharmacokinetic study of high doses of diazepam in schizophrenics.
Authors: Tada K , Moroji T , Sekiguchi R , Motomura H , Noguchi T
Published: January 2020

In this rapid, sensitive method for simultaneously determining diazepam and its biologically active metabolites--nordiazepam, oxazepam, and temazepam--in serum, 250 to 1000 microL of serum is extracted with ether and the extracted compounds are quantified by "high-performance" liquid chromatography on a Shimpack FLC-C8 microparticulate column. Absorbance of the effluent is monitored at 254 nm. The limit of detection in serum is about 10 micrograms/L for each drug. Analytical recovery of each drug added to the serum varied from 91 to 102% for oxazepam and temazepam, from 82 to 99% for nordiazepam and diazepam. Within-day and between-day CVs ranged from 2 to 5% for oxazepam and temazepam, and from 2 to 7% for nordiazepam and diazepam. We also report results on using this assay in a pharmacokinetic study of high doses of diazepam used to treat chronic schizophrenic patients.
Anion-exchange chromatography to determine the concentration of chloride in sweat for diagnosis of cystic fibrosis.
Authors: Miller M E , Cosgriff J M , Schwartz R H
Published: January 2020

For the diagnosis of cystic fibrosis, we use the "Macroduct Sweat Collection System" (Wescor) to sample sweat, and anion-exchange chromatography to determine its chloride content. The chromatographic system consists of a Whatman Partisil SAX 10-microns column, an isocratic phosphate-buffered mobile phase, and ultraviolet detection at 200 nm. The sweat chloride concentrations of 10 individuals with cystic fibrosis ranged from 101 to 131 mmol/L (mean 111, SD 9 mmol/L). For 11 controls the values ranged from 13 to 54 mmol/L (mean 28, SD 11 mmol/L). We conclude that the procedure is reliable for the diagnosis of cystic fibrosis.
University of Virginia case conference. Macroamylase, macro creatine kinase, and other macroenzymes.
Authors: Mifflin T E , Bruns D E , Wrotnoski U , MacMillan R H , Stallings R G , Felder R A , Herold D A
Published: January 2020

The importance of macroenzymes has become increasingly apparent in recent years (1,2). Macroamylase (EC 3.2.1.1) and macro CK (EC 2.7.3.2) are the macroenzymes most commonly noted in the clinical laboratory, and they are frequently responsible for diagnostic confusion. Several methods are available for the confirmation and analysis of macroenzymes, many of which require expensive equipment or complicated techniques. In this report we summarize two cases of macro creatine kinase and two cases of macroamylase that illustrate the clinical importance of recognizing these macroenzymes. We review the features of these macroenzymes, discuss their laboratory evaluation, and describe a simple method that we have used to detect the macroenzymes in these (and other) patients at our institution. Finally, we review the literature on other, less commonly observed macroenzymes in human blood. We stress (a) the impact of methodology on clinical impressions and (b) the importance of discussing laboratory observations with the patient's physician and communicating them to the patient's medical record in writing.
Use of proton nuclear magnetic resonance spectroscopy in detection and study of organic acidurias.
Authors: Iles R A , Hind A J , Chalmers R A
Published: January 2020

We used high-resolution proton nuclear magnetic resonance spectroscopy to detect, identify, and study the major normal and abnormal organic acid metabolites in urine from patients with propionic acidemia, methylmalonic aciduria, branched-chain ketoaciduria, isovaleric acidemia, and glutaric aciduria type I. Characteristic and diagnostic spectra were obtained at 400 MHz for each disorder in all the patients studied and neutral and basic compounds, including amino acids and acylcarnitines, were also detected. The technique is rapid (10 min) and requires small samples (0.5 mL) and no preliminary extraction or derivative preparation. We believe that it is particularly suitable for the rapid and acute diagnosis of inborn errors of metabolism, especially the organic acidurias, and for acute pediatric clinical care, when rapid monitoring of major metabolic alterations is required in a time scale suitable to influence directly and immediately the therapy of the patients concerned.
Measurement of creatine kinase MM sub-type by anion-exchange liquid chromatography.
Authors: Wu A H , Gornet T G
Published: January 2020

Electrophoretic and isoelectric focusing studies have demonstrated the presence of multiple sub-forms of the major creatine kinase isoenzymes, and preliminary work has suggested that measurement of the mm sub-forms provides early diagnostic information concerning acute myocardial infarction. We developed an isocratic method based on liquid chromatography to examine further the clinical potential of measuring these sub-forms. An anion-exchange column is used coupled with the fluorometric detection of NADPH after reaction of the sub-forms with CK reagents, which are added through a post-column pump. We examined various chromatographic variables in producing the best separations. Results for blood collected from normal individuals and patients with acute myocardial infarction and skeletal muscle diseases are compared. We conclude that for routine CK-MM sub-form analysis, this chromatographic procedure is better suited than isoelectric focusing because of the faster turn-around and lower costs.
Direct-calibration method for determination of aluminum in serum is comparable with the protein-precipitation technique.
Authors: Bradley C , Leung F Y , Slavin W , Henderson A R
Published: January 2020

We compared results by two methods for serum Al determination: matrix modification with direct calibration in a stabilized-temperature platform furnace (Clin Chem 28, 2139, 1982) and a technique involving extraction with nitric acid before atomic absorption spectrometry (Clin Chem 30, 1216, 1984). The two methods gave similar results with use of either a deuterium or a Zeeman system of background correction, but gave different slopes for standard additions (mA X s per microgram/L), depending on the Al content of the serum, an effect not seen with aqueous solutions. These differences do not affect the accuracy of the Al determination up to 150 micrograms/L.
In situ monitoring.
Authors: Hicks J M
Published: January 2020

In situ monitoring has been increasingly accepted during the past five years. This approach has been applied to measurement of gas tensions such as pO2 and pCO2, to pH and ions, to the assessment of jaundice by measurement of bilirubin, and to analysis for 14CO2 after administration of labeled antipyrine and aminopyrine, for evaluation of hepatic damage. Various techniques have been developed for in situ monitoring: transcutaneous measurements, implantable sensors, ingestible radio-sensitive pills, and breath analysis. Here I review some of these approaches and their limitations. These limitations include the sparsity of circumstances under which the technique can be applied and the probable increase in health-care costs.
Hypercholinesterasemia with isoenzymic alteration in a family.
Authors: Hada T , Yamawaki M , Moriwaki Y , Tamura S , Yamamoto T , Amuro Y , Nabeshima K , Higashino K
Published: January 2020

A family with hypercholinesterasemia with isoenzymic alteration is reported. The propositus, a 55-year-old woman, was admitted to our hospital because of diabetes mellitus. Because her cholinesterase activity (delta pH 3.2) was supranormal, with no other abnormal liver-function test result throughout the hospitalization period, and was independent of her disease state, we investigated whether this condition might be familial. We studied six of her 17 family members in three generations. All six had above-normal serum cholinesterase activity. Gradient gel electrophoresis on polyacrylamide showed that the normal control individuals had seven isoenzymes, but all the family members with hypercholinesterasemia had two additional isoenzymes. The enzymic properties of the affected members were similar to those of the normal individuals. Hypercholinesterasemia in this family seems to be the result of an increased number of enzyme molecules, but how this isoenzymic alteration emerged remains obscure.
Improved estimation of fructosamine, as a measure of glycated serum protein, with the Technicon RA-1000 analyzer.
Authors: San-Gil F , Schier G M , Moses R G , Gan I E
Published: January 2020

We present an improved fructosamine assay for use with the Technicon RA-1000 analyzer. Features of this assay include: specimen throughput of 100 per hour, within-batch CV of 1.5%, between-batch CV of 2.4%, standard curve linear up to 24.9 mmol of fructosamine per liter, and good correlation (r = 0.82) with hemoglobin A1.
Amplified flow-cytometric separation-free fluorescence immunoassays.
Authors: Saunders G C , Jett J H , Martin J C
Published: January 2020

An equilibrium-type competitive-binding fluorescence immunoassay with high sensitivity and excellent precision is described that obviates separation of free from bound label. In the assay relatively large (10 microns diameter) antibody-coated non-fluorescent particles and very small (0.10 micron diameter) antigen-coated fluorescent latex particles are used. Soluble nonlabeled antigen competes with antigen on the microspheres for antibody binding sites on the larger spheres. After equilibrium is attained, the fluorescence distribution of 5000 of the large spheres is measured in a flow cytometer. The mean values for the fluorescence distribution obtained from samples containing known concentrations of soluble antigen are used to construct a standard displacement curve. In a prototype assay for the antigen horseradish peroxidase, a sensitivity of 10(-12) mol/L has been achieved. Undiluted serum can be assayed without loss of sensitivity. Preliminary experiments also indicate that double-antibody "sandwich"-type assays of very high sensitivity (10(-14) mol/L) are also possible when this dual-sphere concept is exploited.
Single-cuvet sequential determination of triglyceride and cholesterol.
Authors: Zoppi F
Published: January 2020

I describe a sequential method for kinetic assay of serum triglyceride and cholesterol in a single cuvet. Triglyceride is assayed first, by an ultraviolet enzymic method with ATP and NADH as the driving reagents (Clin Chem 21:1627-1629, 1975); cholesterol is then assayed with use of cholesterol oxidase, peroxidase, and the Trinder reaction. Excess NADH left by the triglyceride assay is removed by adding either pyruvate or glycerol to the cholesterol reagent; the addition prevents interference of NADH with nascent H2O2, and does not affect the apparent Km of cholesterol oxidase or the pseudo-first-order reaction of the cholesterol assay. The method, tested in both a random-access analyzer (Hitachi 705) and a batch analyzer (Olli C/D), at three concentrations, gave the following within-run CVs:3.0, 1.6, 1.5% for cholesterol; 4.0, 4.2, 5.4% for triglyceride. The corresponding between-run CVs were 12.5, 9.0, 8.1% and 12.8, 11.1, 8.9%. The calibration was linear to at least 8.0 g/L for both cholesterol and triglyceride. Interference from bilirubin, hemoglobin, and certain drugs is almost negligible. Correlation studies with reference and routine methods showed r values ranging from 0.985 to 0.995 for cholesterol, 0.984 to 0.991 for triglyceride.
Computer evaluation of statistical procedures, and a new quality-control statistical procedure.
Authors: Blum A S
Published: January 2020

I describe a program for definitive comparison of different quality-control statistical procedures. A microcomputer simulates quality-control results generated by repetitive analytical runs. It applies various statistical rules to each result, tabulating rule breaks to evaluate rules as routinely applied by the analyst. The process repeats with increasing amounts of random and systematic error. Rate of false rejection and true error detection for currently popular statistical procedures were comparatively evaluated together with a new multirule procedure described here. The nature of the analyst's response to out-of-control signals was also evaluated. A single-rule protocol that is as effective as the multirule protocol of Westgard et al. (Clin Chem 27:493, 1981) is reported.
Cholesterol determination in serum after fractionation of lipoproteins by immunoprecipitation.
Authors: Heuck C C , Erbe I , Mathias D
Published: January 2020

We investigated the immunoprecipitation of apolipoprotein B-binding lipoproteins, or of apo A-I- and apo C-binding lipoproteins, by delipidated antiserum for measuring cholesterol in the nonprecipitated lipoprotein fractions. After immunoprecipitation of serum with delipidated anti-apo B, we determined by immunoelectrophoresis that no beta- or pre-beta lipoproteins were present, whereas alpha-lipoproteins remained in the supernate. Conversely, after immunoprecipitation with an antiserum against apo A-I + apo C, only lipoproteins with beta-mobility were detected and no apo B from beta-lipoproteins was in the precipitate. The concentration of cholesterol in the supernate after immunoprecipitation with anti-apo B correlated highly (r = 0.93, n = 118) with cholesterol measured after precipitation with phosphotungstic acid/MgCl2. The cholesterol concentration after immunoprecipitation with anti-apo AI and anti-apo C correlated similarly well (r = 0.94, n = 145) with the LDL-cholesterol calculated by Friedewald's formula in serum specimens reflecting moderate hyperlipoproteinemia.
Effect of storage on measurement of ionized calcium in serum of uremic patients.
Authors: Nikolakakis N I , De Francisco A M , Rodger R S , Gaiger E , Goodship T H , Ward M K
Published: January 2020

We studied, in 70 acidotic and non-acidotic uremic patients, the analytical variance in serum ionized calcium as related to duration and temperature of storage. Storage of serum or whole blood at 4 degrees C for as long as 6 h did not significantly alter the measured concentration of ionized calcium in the serum. Storage at room temperature for 6 h, or longer at 4 degrees C or -20 degrees C, resulted in inaccuracies in 39 to 79% of the samples of serum and in 38 to 92% of the samples of whole blood. These errors were not negated by correcting the values for ionized calcium to a pH of 7.40. Indeed, corrected values for calcium were even more unreliable in acidotic patients. We conclude that samples from uremic patients should be analyzed for ionized calcium within 2 h, or within 6 h if stored at 4 degrees C.
Detection of thyroid disorders by use of basal thyrotropin values determined with an optimized "sandwich" enzyme immunoassay.
Authors: Bernutz C , Kewenig M , Horn K , Pickardt C R
Published: January 2020

To measure the concentrations of thyrotropin (thyroid-stimulating hormone), we used the components of a commercially available two-step "sandwich" enzyme immunoassay (Enzymun-Test TSH, Boehringer Mannheim) based on the specific binding of the beta-subunit of thyrotropin by monoclonal antibodies coated on polystyrene tubes. By modifying the original assay protocol, we lowered the limit of detection to 0.18 milli-int. units/L, using a total incubation period of 22 h. With this modification we could differentiate between patients responsive to administration of thyroliberin (thyrotropin-releasing factor) and those who were non-responders, by measuring only the basal concentration of thyrotropin. Furthermore, we demonstrated a correlation between the basal concentration of thyrotropin and its increase after administration of thyroliberin (r = 0.77, n = 48).
Normal "anion gap" (hyperchloremic) acidosis.
Authors: Walmsley R N , White G H
Published: January 2020

Hyperchloremic metabolic acidosis in which the anion gap is within normal limits is a common condition in the hospital population, and often presents a difficult diagnostic problem. We describe nine typical cases of this disorder and suggest a logical approach to its evaluation.
Abnormal lactate dehydrogenase isoenzyme in serum and tumor tissue of a patient with neuroblastoma.
Authors: Otsu N , Hirata M , Miyazawa K , Tuboi S
Published: January 2020

Serum and tumor tissue of a patient with neuroblastoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), which, on agarose gel electrophoresis, migrated between LDH-2 and LDH-3 with a mobility the same as that of the extra LDH isoenzyme found in normal human erythrocytes. On surgical removal of the tumor, the high total LDH activity (775 U/L) in the serum of the patient rapidly decreased to normal (70-220 U/L), and the abnormal LDH isoenzyme was no longer detected. The total LDH activity of the abnormal LDH isoenzyme per gram of hemoglobin in the tumor tissue was 26 times that of erythrocytes, suggesting that the abnormal isoenzyme originated mainly from the tumor cells themselves rather than the erythrocytes contained in the tumor tissue. This first report on the appearance of the abnormal LDH isoenzyme in a patient with neuroblastoma suggests that this abnormal LDH isoenzyme may have some significance as a marker enzyme for neurogenic tumors.
Mixed acid-base disorders.
Authors: Walmsley R N , White G H
Published: January 2020

Mixed acid-base disorders, the occurrence of two or more primary acid-base disturbances in the same patient, are common in the hospital population, but are usually misdiagnosed because of lack of knowledge of the consequences of the primary disturbances. This paper describes seven examples of these disorders recently seen in the authors' hospital, and provides a logical approach to their diagnosis.
Effect of bias in hematofluorometer measurements of protoporphyrin in screening programs for lead poisoning.
Authors: Mitchell D G , Doran D
Published: January 2020

Values for erythrocyte protoporphyrin (EP), measured in our laboratory after extraction with ethyl acetate-acetic acid, were compared with hematofluorometer measurements made in 21 other laboratories. We found that: (a) for samples of patients' blood, hematofluorometer results were 11 to 28% lower than the extraction-based values, depending on the concentration of EP and the mathematical model used; (b) hematofluorometers had mean errors of 0 to 3% for federal proficiency-testing samples; (c) there were no performance differences between fresh and shipped blood for the six laboratories that were analyzing both; (d) a hematofluorometer with a 20% low bias at an EP concentration of 500 micrograms per liter of whole blood (by the extraction method) will not detect about a third of the children whose EP concentration exceeds that cutoff value; and (e) at this same cutoff value for EP, the extraction tests detects about 45% of children whose blood lead exceeds 300 micrograms/L, whereas a 20% low-bias hematofluorometer detects only about 37%.
MM subisoenzymes of creatine kinase as an index of disease activity in polymyositis.
Authors: Annesley T M , Strongwater S L , Schnitzer T J
Published: January 2020

Creatine kinase (CK; EC 2.7.3.2), although the most commonly measured enzyme for assessing disease activity in polymyositis, is not always a reliable indicator of the extent and severity of myositis. Recently, the CK-MM isoenzyme has been found to undergo post-synthetic modifications upon release into the serum, such that electrophoretically identifiable sub-bands or subisoenzymes--MM1, MM2, and MM3--are produced. To determine the diagnostic and discriminative value of these subisoenzymes in polymyositis, we analyzed CK and its MM subisoenzyme forms in serum samples from 22 patients with myositis and from 23 controls. In the presence of inflammatory myositis and increased total CK activity, MM-patterns correlated with the clinical trend, often more accurately than did measurements of total CK. MM1 proportions greater than 30% of total CK-MM or ratios of MM3 to MM1 less than 1 were associated with an improving or stable condition, whereas MM1 activity less than 30% or MM3/MM1 greater than 1 reflected a deteriorating course of disease. Patients whose disease was assessed to be clinically deteriorating were clearly distinguished from patients with improving disease by their subisoenzyme patterns (p less than 0.01). Thus these patterns add significantly to the information obtainable by routine blood analysis.