bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of Catecholamines in Urine by Positive-Ion Electrospray Tandem Mass Spectrometry
Authors: Kushnir Mark M , Urry Francis M , Frank Elizabeth L , Roberts William L , Shushan Bori 
Published: January 2020

Abstract
Background: Determination of urinary catecholamines (CATs) is considered important for clinical diagnosis of pheochromocytoma, paraganglioma, and neuroblastoma. The major disadvantages of existing tests include relatively long instrumental analysis time and potential interference from drugs and drug metabolites that are structurally similar to CATs.
Methods: CATs were extracted from a 300-μL aliquot of urine by a two-step liquid-liquid extraction method specific for compounds containing a catechol group. Chromatographic separation did not require the use of ion-pairing reagents, which typically hinder MS detection but are frequently used in HPLC analysis of CATs. Instrumental analysis was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring mode. Stable-isotope-labeled CATs were used as internal standards.
Results: Epinephrine (E), norepinephrine (NE), and dopamine (D) were measured within 3.5 min instrumental run time. Quantification limits were 2.5 μg/L for E and D and 10 μg/L for NE. The total imprecision (CV) was ≤9.6%; extraction recoveries were 71% ± 12%.
Conclusions: HPLC with ESI-MS/MS in combination with sample preparation specific to catechol group-containing compounds allows rapid testing for disorders associated with increased CAT concentrations. The method is free of interferences from drugs and drug metabolites, which commonly interfere with HPLC methods.
Point-of-Care Time-resolved Immunofluorometric Assay for Human Pregnancy-associated Plasma Protein A: Use in First-Trimester Screening for Down Syndrome
Authors: Qin Qiu-Ping , Christiansen Michael , Pettersson Kim 
Published: January 2020

AbstractBackground: Screening for Down syndrome in the first trimester by a combination of fetal nuchal translucency thickness and maternal serum pregnancy-associated plasma protein A (PAPP-A) and free β-human chorionic gonadotropin has been shown to be effective and efficient. We aimed to develop a fast point-of-care assay that could be placed in one-stop clinics for the measurement of PAPP-A.Methods: We developed a two-site, one-step assay that uses two monoclonal antibodies (mAbs) to PAPP-A, based on a dry-reagent, all-in-one immunoassay concept with a stable fluorescent lanthanide chelate and time-resolved fluorometry. One antibody (mAb 10E1) was biotinylated, and the other (mAb 234-5) was europium-labeled, both via the ε-amino groups of surface lysine residues. The assay was performed on an AIO immunoanalyzer at 36 °C in single, streptavidin-coated microtitration wells that contained the dry reagents. PAPP-A, either in free or complexed form, was detected by the antibodies used.Results: The assay procedure required 20 min and used 10 μL of sample. The calibration curve was linear from 5 to 10 000 mIU/L. The detection limit was 0.5 mIU/L. Intra- and interassay imprecision (CV) was ≤4.3% and 8.3%, respectively, for whole blood, plasma, or serum samples. Recovery was 93–96% for serum, 95–108% for heparin-derived whole blood, and 98–103% for heparin-derived plasma. Parallelism was observed in all three matrices. Results correlated [slope = 0.85 (confidence interval, 0.82–0.87); intercept = −33 (confidence interval, −58 to −9); Sy|x = 85 mIU/L; r = 0.991; n = 100] with those obtained by a Delfia assay. Heparin did not affect the assay, but EDTA markedly reduced PAPP-A values. PAPP-A was stable at 4 °C for at least 18 days in serum and for 8 days in heparin-derived whole blood or plasma.Conclusions: The present assay appears suited for use in one-stop clinics for screening for Down syndrome in the first trimester, with results available within 1 h.
Performance of Four Homogeneous Direct Methods for LDL-Cholesterol
Authors: Miller W Greg , Waymack Parvin P , Anderson F Philip , Ethridge Steven F , Jayne Eduviges C 
Published: January 2020

AbstractBackground: Homogeneous LDL-cholesterol methods from Genzyme, Reference Diagnostics, Roche, and Sigma were evaluated for precision, accuracy, and specificity for LDL in the presence of abnormal lipoproteins.Methods: Each homogeneous method was performed by a Roche/Hitachi 911 according to the vendors’ instructions, and the results were compared with the β-quantification reference method. We measured precision over 20 days using quality-control and frozen serum specimens. Sera from 100 study participants, including 60 with hyperlipidemias, were assayed by each method. Accuracy was evaluated from regression and total error analysis. Specificity was evaluated from the bias (as a percentage) vs concentration of triglycerides.Results: The total CV was &lt;2% for all methods. Regression slope and intercept (with 95% confidence intervals) were as follows: Genzyme, 0.955 (0.92 to 0.99) and 30.3 (−12 to 73) mg/L; Reference Diagnostics, 0.975 (0.93 to 1.02) and −8 (−63 to 47) mg/L; Roche, 1.067 (1.02 to 1.11) and −101 (−161 to −42) mg/L; and Sigma, 0.964 (0.91 to 1.02) and 164 (89 to 239) mg/L. The percentages of individual results with &gt;12% bias were as follows: Genzyme, 8.0%; Reference Diagnostics, 11.0%; Roche, 10.0%; and Sigma, 30.0%. Total error calculated from mean systematic bias and all-sources random bias was as follows: Genzyme, 12.6%; Reference Diagnostics, 16.5%; Roche, 41.6%; and Sigma, 38.3%. Slopes of bias (as a percentage) vs triglycerides were P &lt;0.001 for all methods except the Roche method, which was P = 0.094.Conclusions: The evaluated methods show nonspecificity toward abnormal lipoproteins, thus compromising their ability to satisfy the National Cholesterol Education Program goal for a total error of &lt;12%. These homogeneous LDL-cholesterol results do not improve on the performance of LDL-cholesterol calculated by the Friedewald equation at triglyceride concentrations &lt;4000 mg/L.
Immunometric Assay Interference
Authors: Bjerner Johan , Nustad Kjell , Norum Lars F , Olsen Kari Hauge , Børmer Ole P 
Published: January 2020

AbstractBackground: The primary aim of the study was to reduce interference in an in-house two-site, two-step immunometric assay.Methods: In the running laboratory routine, 11 261 samples were tested with a carcinoembryonic antigen (CEA) assay with bovine immunoglobulin but no murine immunoglobulins in the buffer, in parallel to our routine CEA assay, using 15 mg/L heat-treated nonspecific murine immunoglobulin (MAK33) in the buffer and with the Fc fragments removed from the capture antibody.Results: The frequency of interference was estimated to be 4.0% (95% confidence interval, 3.3–4.7%). The addition of 15 mg/L native MAK33 had little effect (frequency, 3.9%; 95% confidence interval, 3.2–4.6%), whereas adding 15 mg/L heat-treated MAK33 reduced interference to 0.86% (0.61–1.12%), and adding 50 mg/L reduced it further to 0.06% (0–0.13%). Removing the Fc fragments by itself reduced interference to 0.10% (0.02–0.19%). There were no statistically significant differences for age (P &lt;0.23) or gender (P &lt;0.40) between patients with interference (n = 210) and a randomly selected interference-negative control group (n = 186). Interference was not constant in patients: 15 of 25 individuals positive for interference and with four or more samples screened for interference had an interference-negative sample either before or after the peak of interference.Conclusions: In a two-site, two-step immunometric assay using mouse monoclonal antibodies, use of heat-treated nonspecific murine immunoglobulin in the buffer or removal of the Fc fragment from the capture antibody could improve performance.
Candidate Reference Method for Total Thyroxine in Human Serum
Authors: Tai Susan S-C , Sniegoski Lorna T , Welch Michael J 
Published: January 2020

Abstract
Background: There is a need for a critically evaluated reference method for thyroxine to provide an accuracy base to which routine methods can be traceable. We describe a candidate reference method involving isotope-dilution coupled with liquid chromatography/mass spectrometry.
Methods: An isotopically labeled internal standard, thyroxine-d5, was added to serum, followed by equilibration, protein precipitation, and ethyl acetate and solid-phase extractions to prepare samples for liquid chromatography-mass spectrometry electrospray ionization (LC/MS-ESI) analysis. For separation, a Zorbax Eclipse XDB-C18 column was used with a mobile phase consisting of 1 mL/L acetic acid in acetonitrile-water (32:68 by volume) for positive ions and a Zorbax Extend-C18 column with a mobile phase consisting of 2 mL/L ammonium hydroxide in methanol-water (32:68 by volume) for negative ions. [M + H]+ ions at m/z 778 and 783 for thyroxine and its labeled internal standard were monitored for positive ions and [M − H]− ions at m/z 776 and 781 for negative ions. Samples of frozen serum pools were prepared and measured in three separate sets.
Results: Within-set CVs were 0.2–1.0%. The correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) were 0.999–1.000. Positive- and negative-ion measurements agreed with a mean difference of 0.45% at three concentrations (50, 110, and 168 μg/L). The detection limits (at a signal-to-noise ratio of ∼3 to 5) were 30 and 20 pg for positive and negative ions, respectively. The results from the LC/MS-ESI method were within 1 SD of the composite means from many routine clinical methods, although it appears that the clinical method means may be biased high by 4–5 μg/L across the concentrations. Some routine clinical methods may be biased by up to 20% at low concentrations.
Conclusions: This well-characterized LC/MS-ESI method for total serum thyroxine with a theoretically sound approach, demonstrated good accuracy and precision, and low susceptibility to interferences qualifies as a candidate reference method. Use of this reference method as an accuracy base may reduce the apparent biases in routine methods along with the high interlaboratory imprecision.
Use of Constant Denaturant Capillary Electrophoresis of Pooled Blood Samples to Identify Single-Nucleotide Polymorphisms in the Genes (Scnn1a and Scnn1b) Encoding the α and β Subunits of the Epithelial Sodium Channel
Authors: Xue Ming-Zhan , Bonny Olivier , Morgenthaler Stephan , Bochud Murielle , Mooser Vincent , Thilly William G , Schild Laurent , Leong-Morgenthaler Phaik-Mooi 
Published: January 2020

Abstract
Background: The epithelial sodium channel (ENaC) is composed of three homologous subunits: α, β, and γ. Mutations in the Scnn1b and Scnn1g genes, which encode the β and the γ subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the α subunit, has not been investigated.
Methods: We screened for mutations in the COOH termini of the α and β subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of α- and β-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples.
Results: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of α-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G→T and 1987A→G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of β-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C→T (T594M).
Conclusion: CDCE rapidly detects point mutations in these candidate disease genes.
Blood Glutathione Disulfide: In Vivo Factor or in Vitro Artifact?
Authors: Rossi Ranieri , Milzani Aldo , Dalle-Donne Isabella , Giustarini Daniela , Lusini Lorenzo , Colombo Roberto , Di Simplicio Paolo 
Published: January 2020

AbstractBackground: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to &gt;500 μmol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG.Methods: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector.Results: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30–150 μmol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide.Conclusions: GSSG is present in healthy human blood at low concentrations (2–6 μmol/L), and most published data on GSSG may be affected by artifacts.
Improved Method for Gas Chromatographic–Mass Spectrometric Analysis of 1-13C-labeled Long-Chain Fatty Acids in Plasma Samples
Authors: Hernández-Pérez José M , Cabré Eduard , Fluvià Lourdes , Motos Ágata , Pastor Cruz , Corominas August , Gassull Miquel A 
Published: January 2020

Abstract
Background: Gas chromatographic–mass spectrometric (GC/MS) tracking of stable-isotope-labeled substrates is useful in metabolic studies. However, GC/MS analysis of long-chain fatty acid methyl esters yields results that mostly depend on their concentration in the system. We describe a protocol aimed to obviate this and other drawbacks in plasma [1-13C]palmitic and [1-13C]oleic acid measurements.
Methods: Lipoproteins were separated by sequential ultracentrifugation. Free or esterified heptadecanoic acid was used as internal standard. Fatty acids were derivatized to trimethylsilyl (TMS) esters. GC separation was in isothermal mode at 210 °C for 27 min. For both TMS-palmitate and TMS-oleate, M and [M + 1] signals were simultaneously acquired with a dual acquisition program in single-ion monitoring mode. Calibration mixtures containing increasing amounts of labeled fatty acids were prepared gravimetrically to construct calibration curves for isotopic enrichment. Likewise, five calibration curves (for increasing concentrations) were constructed for each fatty acid; this allowed selection of the most appropriate curve for the concentration in a plasma sample.
Results: Oleic acid-TMS ester was clearly separated from that of its stereoisomer, elaidic acid. Within a 10-fold concentration range, the isotopic ratio was independent on the amount of the analyte in the sample, with a maximum uncertainty of 0.34% in terms of molar percent excess. In addition, the within- and between-day imprecision (CV) of the method was &lt;1%.
Conclusion: Results obtained with this method are independent of concentration and sufficiently precise for tracking 1-13C-labeled palmitic and oleic acids in biological samples
Determination of Sulpiride by Capillary Electrophoresis with End-Column Electrogenerated Chemiluminescence Detection
Authors: Liu Jifeng , Cao Weidong , Qiu Haibo , Sun Xiuhua , Yang Xiurong , Wang Erkang 
Published: January 2020

Abstract
Background: Capillary electrophoresis (CE) with tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)32+]-electrogenerated chemiluminescence (ECL) detection is a promising method for clinical analysis. In this study, a method combining CE with Ru(bpy)32+ ECL (CE-ECL) detection that can be applied to amine-containing clinical species was developed, and the performance of CE-ECL as a quantitative method for determination of sulpiride in human plasma or urine was evaluated.
Methods: Sulpiride was separated by capillary zone electrophoresis in uncoated fused-silica capillaries [50 cm × 25 μm (i.d.)] filled with phosphate buffer (pH 8.0) and a driving voltage of +15 kV, with end-column Ru(bpy)32+ ECL detection. A platinum disc electrode was used as working electrode. Sulpiride in human plasma or urine samples (100 μL) was extracted by a double-step liquid-liquid extraction procedure, dried under nitrogen at 35 °C in a water bath, and reconstituted with 100 μL of filtered water. The extraction solvent was ethyl acetate–dichloromethane (5:1 by volume).
Results: Under optimum conditions (pH 8.0 phosphate buffer, injection for 6 s at 10 kV, and +1.2 V as detection potential), separation of sulpiride was accomplished within 4 min. The calibration curve was linear over a concentration range of 0.05–25.0 μmol/L, and the limit of detection was 2.9 × 10−8 mol/L for sulpiride. Intra- and interday CVs for ECL intensities were &lt;6%. Extraction recoveries of sulpiride were 95.6–101% with CVs of 2.9–6.0%. The method was clinically validated for patient plasma and urine samples.
Conclusions: CE combined with Ru(bpy)32+ ECL is reproducible, precise, selective, and enables the analysis of sulpiride in human plasma and urine. It thus is of value for rapid and efficient analysis of amine-containing analytes of clinical interest.
Practice Guidelines for Tumor Marker Use in the Clinic
Authors: Sturgeon Catharine 
Published: January 2020

AbstractBackground: Increasing interest in implementing the practice of evidence-based medicine in oncology has encouraged the development of clinical guidelines, many of which include recommendations about the appropriate use of serum tumor markers.Methods: Recent national and international guidelines relating to the use of tumor markers in germ cell, colorectal, breast, ovarian, prostate, lung, neuroendocrine, and thyroid cancers were identified from the scientific literature and other sources and tabulated.Results: Guideline recommendations developed by national and international groups and relating to the use of tumor markers for specific cancers are reviewed and compared, considering the recommendations made for their use in screening, diagnosis, prognosis, and monitoring of therapy. Potential advantages and disadvantages of clinical guidelines, how best to implement them, and means of auditing their effectiveness are also considered.Conclusions: Excellent clinical guidelines, including recommendations for the most appropriate use of tumor markers, are already available for many cancers. Many questions relating to optimal use of these important tests remain to be answered, but current guidelines already contain much valuable information and advice. Further dissemination and implementation of the guidelines should encourage better use of tumor markers in clinical practice. Careful audit studies are also required to establish the impact of these guidelines on the practice of evidence-based medicine.
Tumor-associated Trypsin Inhibitor
Authors: Stenman Ulf-Håkan 
Published: January 2020

AbstractTumor-associated trypsin inhibitor (TATI) is a low-molecular-weight (6 kDa) trypsin inhibitor that has been used as a marker for ovarian cancer. It is also expressed together with tumor-associated trypsin by many other tumors, and increased serum concentrations of TATI occur in connection with these. TATI is a prognostic marker for ovarian, bladder, and kidney cancer, which may be associated with the participation of trypsin in protease cascades contributing to tumor invasiveness.
Presence of Filterable and Nonfilterable mRNA in the Plasma of Cancer Patients and Healthy Individuals
Authors: Ng Enders KO , Tsui Nancy BY , Lam Nicole YL , Chiu Rossa WK , Yu Simon CH , Wong SC Cesar , Lo Elena SF , Rainer Timothy H , Johnson Philip J , Lo YM Dennis 
Published: January 2020

AbstractBackground: As RNA is labile, we investigated whether circulating RNA in human plasma may be present in a particle-associated form.Methods: Blood was collected from 27 healthy individuals and 16 hepatocellular carcinoma (HCC) patients. The plasma from each individual was processed by two means: filtration through filters with different pore sizes (from 5 μm to 0.22 μm) and ultracentrifugation. We assessed plasma RNA content by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts and plasma DNA by a real-time quantitative PCR assay for the β-globin gene.Results: The plasma GAPDH mRNA concentrations in the healthy individuals were significantly different in every pair of these filter sizes (P &lt;0.05 for each pair). Overall, the plasma GAPDH mRNA concentration was higher by a median of 15-fold (interquartile range, 10- to 24-fold) in the paired unfiltered sample than in the sample filtered through a 0.22 μm filter. In contrast, no significant difference was seen in β-globin DNA concentrations among different pore-size-filtered plasma samples (P = 0.455). Similarly, a significant difference was observed for RNA, but not DNA, between unfiltered plasma and ultracentrifuged plasma (P &lt;0.05). No significant difference in GAPDH mRNA concentrations was seen between the 0.22-μm-filtered plasma samples and the ultracentrifuged plasma samples (P &gt;0.05). In HCC patients, filtration with a 0.22 μm filter produced a median 9.3-fold (interquartile range, 6.9- to 311-fold) reduction in GAPDH mRNA concentration in plasma. Plasma GAPDH mRNA concentrations in HCC patients were significantly higher than those in healthy individuals, both with or without filtration (P &lt;0.0 5 for filtered plasma samples; P &lt;0.005 for unfiltered plasma samples).Conclusions: A substantial proportion of plasma mRNA species is particle-associated. In HCC patients, both circulating particle- and non-particle-associated plasma RNA are increased.