bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of Coumarin-type Anticoagulants in Human Plasma by HPLC-Electrospray Ionization Tandem Mass Spectrometry with an Ion Trap Detector
Authors: Kollroser Manfred , Schober Caroline 
Published: January 2020

Abstract
Background: Coumarin-type anticoagulants are used for the long-term treatment and prevention of thromboembolic disorders. The identification of these drugs is crucial in patients with an increased prothrombin time of unknown origin. The aim of this study was to develop a sensitive and specific method for the simultaneous determination of phenprocoumon, acenocoumarol, and warfarin in human plasma by HPLC-electrospray ionization tandem mass spectrometry.
Methods: After addition of the internal standard, p-chlorowarfarin, plasma samples were extracted using Oasis® MCX solid-phase extraction cartridges. The compounds were separated on a Symmetry C18 column (Waters) with a mobile phase of acetonitrile–1 g/L formic acid (75:25 by volume) at a flow rate of 0.5 mL/min.
Results: Extraction and separation of the three drugs and the internal standard were accomplished in 9 min. The overall extraction efficiency was &gt;89% for all three compounds. The limits of detection were 1 μg/L for phenprocoumon and warfarin and 10 μg/L for acenocoumarol. Regression analysis of the calibration data revealed good correlation (r2 ≥0.995) for all compounds. Within-run accuracies for quality-control samples were ± 1% to 7% of the target concentration, with CVs &lt;9%.
Conclusions: The proposed method enables the unambiguous identification and quantification of phenprocoumon, warfarin, and acenocoumarol in both clinical and forensic specimens. This method combines a new, rapid solid-phase extraction procedure with an extremely fast chromatographic analysis, which is especially advantageous for clinical laboratories.
Polymorphisms of TLR4: Rapid Genotyping and Reduced Response to Lipopolysaccharide of TLR4 Mutant Alleles
Authors: Schmitt Christopher , Humeny Andreas , Becker Cord-Michael , Brune Kay , Pahl Andreas 
Published: January 2020

AbstractBackground: Pathogen recognition receptors such as Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, lead to the activation of innate immunity. Genetic variations in these receptors may lead to an altered host immune response to pathogens.Methods: We developed homogeneous fluorescence-based PCR assays as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) genotyping assays to detect TLR4 polymorphisms. These assays were compared with restriction fragment length polymorphism (RFLP) analysis. Peripheral blood monocytes from donors with differing genotypes were prepared and exposed to bacterial products in vitro. The abundance of mRNAs of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α from these monocytes were monitored by real-time reverse transcription-PCR.Results: By our homogeneous PCR method, the allele frequencies were 5.6% for the TLR4 Asp299Gly and 6.0% for the TLR4 Thr399Ile polymorphism in 116 healthy German Caucasians. Nine incorrect genotype calls were detected in the RFLP analysis and two in the TaqMan genotype analysis. MALDI-TOF-MS allowed clear detection of all TLR4 alleles. Monocytes from donors homozygous for the TLR4 mutant alleles Asp299Gly and Thr399Ile were lipopolysaccharide hyporesponsive and exhibited median effective concentrations (EC50s) approximately fourfold higher than those of monocytes carrying wild-type or heterozygous alleles. In contrast, a TLR2 agonist elicited similar responses in monocytes irrespective of the TLR4 genotype.Conclusions: Homogeneous fluorescence-based PCR assays provide a specific and sensitive method for high-throughput genotyping of TLR4 mutations. The newly developed PCR and MALDI-TOF-MS assays may be useful to evaluate the presence of TLR4 polymorphisms in patients to predict susceptibility to bacterial infection.
European Concerted Action on Anticoagulation. Evaluation of a Method for International Sensitivity Index Calibration of Two Point-of-Care Prothrombin Time (PT) Monitoring Systems (CoaguChek Mini and TAS PT-NC) with Fresh Plasmas Based on Whole-Blood Equivalent PT
Authors: Poller Leon , Keown Michelle , Chauhan Nikhil , van den Besselaar Anton MHP , Tripodi Armando , Shiach Caroline , Jespersen Jorgen 
Published: January 2020

Abstract
Background: The International Sensitivity Index (ISI) calibration of whole-blood prothrombin time (PT) monitors for point-of-care testing (POCT) described by Tripodi et al. (Thromb Haemost 1993;70:921–4) has been shown to be dependable but is too complex and demanding. The use of plasma would simplify calibration of whole-blood POCT PT monitors, but important differences may exist between the ISI for whole blood and plasma calibrations.
Methods: In a 10-center calibration study of two POCT whole-blood monitoring systems (CoaguChek Mini and TAS PT-NC), we characterized the relationship between the log PT for whole blood and fresh plasma with use of single lots of test strips/cards. This relationship (linear) was used to correct the difference between the whole-blood and plasma ISI. The reliability of the correction with different lots of test strips/cards was assessed at three centers. The linear relationship was used to correct the difference in the whole-blood and plasma ISI with four other lots of TAS PT-NC cards and with two additional lots of CoaguChek Mini test strips.
Results: The correction decreased the ISI difference from 13.3% to 0.9% for the TAS PT-NC and from 5.7% to 0.6% for the CoaguChek Mini. In assessments at three centers, which included different lots of test strips/cards, the mean ISI difference was markedly decreased with the TAS PT-NC but not with the CoaguChek Mini, for which the mean ISI difference increased slightly.
Conclusions: The proposed correction resolves the discrepancy between whole-blood and fresh plasma ISI calibrations with TAS PT-NC test cards. The CoaguChek Mini systems could be calibrated without this correction.
Development and Evaluation of an ELISA for Human Trefoil Factor 3
Authors: Vestergaard Else Marie , Poulsen Steen Seier , Grønbæk Henning , Larsen Rune , Nielsen Anette Mertz , Ejskjær Kirsten , Clausen Jes Thorn , Thim Lars , Nexø Ebba 
Published: January 2020

AbstractBackground: The three trefoil factors (TFF1, TFF2, and TFF3) are small peptides believed to cross-link mucous glycoproteins and to play a role in the maintenance and repair of the gastrointestinal mucosa. To define the physiologic and potential diagnostic values of TFF3, assays able to measure TFF3 are warranted.Methods: An ELISA was developed that uses two antibodies from rabbits immunized with recombinant human TFF3 and a calibrator (3–100 pmol/L) prepared from recombinant human TFF3.Results: The ELISA had a detection limit of 3.0 pmol/L. The imprecision (CV) was 5–9% for mean concentrations of 13–65 pmol/L, corresponding to serum concentrations of 65–330 pmol/L. There was no cross-reaction toward human TFF1 and TFF2 (40 nmol/L). Neither food intake nor the menstrual cycle influenced the values of TFF3 significantly. The central 95% reference interval for TFF3 in serum from healthy blood donors (n = 300) was 91–250 pmol/L and showed no variation with age and limited variation with sex. TFF3 was increased in serum from patients (n = 12) with inflammation and/or ulceration of the upper gastrointestinal tract (P &lt;0.05), whereas in serial measurements of serum from three patients with severe exacerbation of chronic inflammatory bowel disease restricted to the colon, normal concentrations and only minor variations during treatment and tapering were observed.Conclusions: The ELISA measures TFF3 in human serum and represents a specific and precise method for measurement of TFF3, which will be of value for further studies of TFF3 in health and disease.
Therapeutic Drug Monitoring of Anticonvulsant Drugs by Micellar HPLC with Direct Injection of Serum Samples
Authors: Martinavarro-Domínguez Adrián , Capella-Peiró Maria-Elisa , Gil-Agustí Mayte , Marcos-Tomás José V , Esteve-Romero Josep 
Published: January 2020

Abstract
Background: We developed a micellar liquid chromatographic (MLC) procedure for the determination of three extensively monitored antiepileptics in serum samples: carbamazepine, phenobarbital, and phenytoin.
Methods: We determined the composition of the mobile phase after modeling the elution behavior of the antiepileptics in hybrid micellar mobile phases of sodium dodecyl sulfate (SDS) with different organic modifiers (propanol, butanol, or pentanol) in an experimental design that used five mobile phases, a C18 column, and ultraviolet detection. In the micellar chromatographic system, the serum samples can be injected directly.
Results: The optimum mobile phase was 70 mL/L butanol in 0.05 mol/L SDS, pH 7, in which the three antiepileptics were resolved in &lt;10 min. Intra- and interday precision was evaluated at four different drug concentrations within the therapeutic range (n =10); CVs were &lt;2.1%. The method was applied to the analysis of 120 serum samples, and results were similar to those obtained by the TDx® method.
Conclusions: The MLC method allows chromatographic determination of three antiepileptics, using an interpretative strategy of optimization, without pretreatment of the serum samples and with direct injection in a hybrid micellar mobile phase of SDS–butanol. The method provides complete resolution and quantification of mixtures of two and three antiepileptics.
Antibodies Directed against the E Region of Pro-Insulin-like Growth Factor-II Used to Evaluate Non-Islet Cell Tumor-induced Hypoglycemia
Authors: van Doorn Jaap , Hoogerbrugge Cok M , Koster Johanna G , Bloemen Ruud J , Hoekman Klaas , Mudde Aart H , van Buul-Offers Sylvia C 
Published: January 2020

AbstractBackground: Detection of incompletely processed precursor forms of insulin-like growth factor-II (“big” IGF-II) in plasma is essential for both the diagnosis and follow-up of non-islet cell tumor-induced hypoglycemia (NICTH) and may be relevant to other diseases as well. RIA using an antibody raised against a synthetic peptide consisting of the first 21 amino acids of the E domain [E(68–88)] of human pro-IGF-II cannot distinguish between E-peptide-containing big IGF-II and cleaved E domain or fragments. We therefore developed and validated an ELISA that specifically detects big IGF-II in plasma.Methods: The ELISA used a solid-phase antibody to E(68–88) and a liquid-phase monoclonal hIGF-II antibody. Pro-IGF-II purified from normal human plasma was used as a calibrator. Acid Sep-Pak C18 extracts of plasma from NICTH patients were analyzed, and the results were compared with those obtained for plasma samples from healthy individuals. In addition, blood specimens derived from dialyzed patients with chronic renal failure, which contained relatively high concentrations of cleaved E domain or fragments, were studied. The results were validated by acid Sephadex G-50 gel filtration.Results: Results from this ELISA indicated that the concentration of big IGF-II in NICTH plasma was higher (mean ± SD, 22.6 ± 9.4 nmol/L) than in normal plasma (3.8 nmol/L). Conversely, the concentrations in pooled CRF plasma (2.0 ± 0.8 nmol/L) were low. Antibodies directed against either E(68–88) or E(13–134) of pro-IGF-II could be used to detect these peptides in tumor tissue by immunohistochemistry.Conclusions: The possibility of quantifying pro-IGF-II by ELISA in plasma represents a potentially useful tool for the diagnosis and follow-up of NICTH and should facilitate further in vitro and in vivo studies on its regulation and function in humans.
Holo-Transcobalamin Is an Early Marker of Changes in Cobalamin Homeostasis. A Randomized Placebo-controlled Study
Authors: Nexo Ebba , Hvas Anne-Mette , Bleie Øyvind , Refsum Helga , Fedosov Sergey N , Vollset Stein Emil , Schneede Jorn , Nordrehaug Jan Erik , Ueland Per Magne , Nygard Ottar Kjell 
Published: January 2020

Abstract
Background: We examined the effect of oral vitamin B12 treatment on fluctuations in plasma total cobalamin and its binding proteins transcobalamin (TC) and haptocorrin (HC).
Methods: Patients (n = 88; age range, 38–80 years) undergoing coronary angiography (part of the homocysteine-lowering Western Norway B-Vitamin Intervention Trial) were allocated to daily oral treatment with (a) vitamin B12 (0.4 mg), folic acid (0.8 mg), and vitamin B6 (40 mg); (b) vitamin B12 and folic acid; (c) vitamin B6; or (d) placebo. EDTA blood was obtained before treatment and 3, 14, 28, and 84 days thereafter.
Results: The intraindividual variation for patients not treated with B12 was ∼10% for plasma total cobalamin, total TC, apo-TC, and apo-HC, and &lt;20% for holo-TC and TC saturation. In B12-treated patients, the maximum change in concentrations was observed already after 3 days for total TC (−16%), holo-TC (+54%), and TC saturation (+82%). At this time holo-HC (+20%) and plasma total cobalamin (+28%) showed an initial burst, but had increased further at 84 days. All changes were highly significant compared with the control group (P &lt;0.0001).
Conclusions: Oral vitamin B12 treatment produces maximal effects on total TC, holo-TC, and TC saturation within 3 days, whereas maximal increases in holo-HC and plasma total cobalamin occur later. The results support the view that holo-TC is an early marker of changes in cobalamin homeostasis.
Guanidinoacetate and Creatine plus Creatinine Assessment in Physiologic Fluids: An Effective Diagnostic Tool for the Biochemical Diagnosis of Arginine:Glycine Amidinotransferase and Guanidinoacetate Methyltransferase Deficiencies
Authors: Carducci Claudia , Birarelli Maurizio , Leuzzi Vincenzo , Carducci Carla , Battini Roberta , Cioni Giovanni , Antonozzi Italo 
Published: January 2020

Abstract
Background: Disorders of creatine metabolism arise from genetic alterations of arginine:glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and the creatine transporter. We developed a strategy for the detection of AGAT and GAMT defects by measurement of guanidinoacetate (GAA) and creatine plus creatinine (Cr+Crn) in biological fluids.
Methods: Three patients with AGAT deficiency from the same pedigree and their eight relatives, as well as a patient affected by a GAMT defect and his parents were analyzed by a new HPLC procedure in comparison with 90 controls. The method, which uses precolumn derivatization with benzoin, separation with a reversed-phase column, and fluorescence detection, has shown good precision and sensitivity and requires minimal sample handling.
Results: In the three AGAT patients, plasma GAA was 0.01–0.04 μmol/L [mean (SD) for neurologically normal controls was 1.16 (0.59) μmol/L], Cr+Crn was 15–29 μmol/L [reference limit in our laboratory, 79 (38) μmol/L]. Urinary GAA was 2.4–5.8 μmol/L [reference, 311 (191) μmol/L], and Cr+Crn was 2.1–3.3 mmol/L [reference, 9.9 (4.1) mmol/L]. We found a smaller decrease in GAA and Cr+Crn in some carriers of an AGAT defect. In the patient with GAMT deficiency, plasma and urine GAA was increased (18.6 and 1783 μmol/L, respectively), and Cr+Crn was decreased in plasma (10.7 μmol/L) and urine (2.1 mmol/L). GAA was increased in the parents’ plasmas and in the mother’s urine.
Conclusion: The assessment of GAA is a new tool for the detection of both GAMT and AGAT deficiencies.
Comparison of Logistic Regression and Neural Net Modeling for Prediction of Prostate Cancer Pathologic Stage
Authors: Veltri Robert W , Chaudhari Manisha , Miller M Craig , Poole Edward C , O’Dowd Gerard J , Partin Alan W 
Published: January 2020

Abstract
Background: Prostate cancer (PCa) pathologic staging remains a challenge for the physician using individual pretreatment variables. We have previously reported that UroScoreTM, a logistic regression (LR)-derived algorithm, can correctly predict organ-confined (OC) disease state with &gt;90% accuracy. This study compares statistical and neural network (NN) approaches to predict PCa stage.
Methods: A subset (756 of 817) of radical prostatectomy patients was assessed: 434 with OC disease, 173 with capsular penetration (NOC-CP), and 149 with metastases (NOC-AD) in the training sample. Additionally, an OC + NOC-CP (n = 607) vs NOC-AD (n = 149) two-outcome model was prepared. Validation sets included 120 or 397 cases not used for modeling. Input variables included clinical and several quantitative biopsy pathology variables. The classification accuracies achieved with a NN with an error back-propagation architecture were compared with those of LR statistical modeling.
Results: We demonstrated &gt;95% detection of OC PCa in three-outcome models, using both computational approaches. For training patient samples that were equally distributed for the three-outcome models, NNs gave a significantly higher overall classification accuracy than the LR approach (40% vs 96%, respectively). In the two-outcome models using either unequal or equal case distribution, the NNs had only a marginal advantage in classification accuracy over LR.
Conclusions: The strength of a mathematics-based disease-outcome model depends on the quality of the input variables, quantity of cases, case sample input distribution, and computational methods of data processing of inputs and outputs. We identified specific advantages for NNs, especially in the prediction of multiple-outcome models, related to the ability to pre- and postprocess inputs and outputs.
Differential Reactivity of Two Homogeneous LDL-Cholesterol Methods to LDL and VLDL Subfractions, as Demonstrated by Ultracentrifugation and HPLC
Authors: Usui Shinichi , Kakuuchi Hajime , Okamoto Motoi , Mizukami Yuki , Okazaki Mitsuyo 
Published: January 2020

AbstractBackground: The analytical and clinical performance of homogeneous LDL-cholesterol assays has been reported, but their reactions with subfractions of LDL and VLDL have not been described in detail.Methods: We evaluated reaction selectivity of two homogeneous LDL-cholesterol assays, LDLk (Kyowa Medex) and LDLd (Daiichi Pure Chemical), with ultracentrifugally isolated VLDL and LDL subfractions to identify the lipoprotein particles from which the cholesterol recognized by these assays originates.Results: The LDLd (y) and LDLk (x) methods correlated highly for whole serum samples: y = 0.986x − 39.5 mg/L (r = 0.966; n = 34). In isolated VLDL, the LDLk and the LDLd methods recovered 17.3% and 23.8% of cholesterol, respectively; but correlation analysis revealed differential reactivity to small and large VLDL particles. For the isolated LDL subfraction of density 1.019–1.040 kg/L, the LDLd method had significantly higher reactivity (95.6–98.7%) than the LDLk (88.4–92.0%). Both methods, however, demonstrated poor recovery (∼50%) for the 1.050–1.063 kg/L fraction, indicating incomplete reactivity with small, dense LDL. Reactivity with lipoprotein(a) was better (71.2–90.8%) for both methods than with small LDL. For intermediate-density lipoprotein (IDL), there was no significant difference in recovery between the two methods (71.7% for LDLk and 68.9% for LDLd), but the LDLk method appeared to be more sensitive to IDL particle size.Conclusions: The two homogeneous assays for LDL-cholesterol demonstrate only partial reactivity to small, dense LDL and nonspecific reactions to VLDL particles. Modification will be required in the homogeneous methods to obtain LDL-cholesterol values equivalent to those obtained by ultracentrifugation.
Lack of Association between Increased Carotid Intima-Media Thickening and Decreased HDL-Cholesterol in a Family with a Novel ABCA1 Variant, G2265T
Authors: Hong Seung Ho , Riley Ward , Rhyne Jeffrey , Friel Gina , Miller Michael 
Published: January 2020

AbstractLow HDL-cholesterol (HDL-C) concentrations are inversely correlated with cardiovascular disease, and previous studies have demonstrated that variants in the ATP-binding cassette transporter, ABCA1, are responsible for a proportion of HDL-C deficiency states. We identified a novel variant in ABCA1 in a kindred with decreased HDL-C. This variant was not identified in &gt;200 chromosomes of healthy individuals. The proband, a heterozygote for G2265T, developed premature coronary artery disease. In addition to low HDL-C, six biological family members heterozygous for the ABCA1 variant exhibited low HDL-C concentrations compared with unaffected family members (0.83 ± 0.32 vs 1.33 ± 0.36 mmol/L; P = 0.009). Despite the decreased HDL-C, carotid artery B-mode ultrasound studies failed to reveal increased intima-media thickening in affected individuals compared with age- and sex-matched controls. Although these data extend previous observations that a single defective ABCA1 allele may lead to decreased HDL-C, associated evidence of early atherosclerosis was not confirmed.
Epitope Mapping of Antibodies against Prostate-specific Antigen with Use of Peptide Libraries
Authors: Leinonen Jari , Wu Ping , Stenman Ulf-Håkan 
Published: January 2020

AbstractBackground: Prostate-specific antigen (PSA) is the most important marker for prostate cancer, but PSA concentrations determined by various assays can differ significantly because of differences in specificity of the antibodies used. To identify epitopes recognized by various monoclonal antibodies (MAbs) to PSA, we have isolated peptides that react with the paratopes of these.Methods: Six anti-PSA MAbs representing three major epitope groups were screened with five cyclic phage display peptide libraries. After selection, the peptide sequences were determined by sequencing of the relevant part of viral DNA. Binding of the phage peptides to the MAbs was monitored by immunoassay.Results: For each MAb, several paratope-binding peptides with distinct sequence motifs were identified, but only ∼10% showed similarity with the PSA sequence. Some of these correctly predicted the location of the epitopes. By sequential panning of the library with two closely related MAbs, we identified peptides reacting equally with both MAbs. When analyzed against a large panel of PSA MAbs, the peptides generally showed restricted specificity toward the MAb used for selection, but some peptides bound to several related MAbs.Conclusions: Most of the cyclic peptides selected with PSA MAbs are specific for the MAb used for selection and do not resemble any sequence on the antigen. Peptides reactive with two MAbs recognizing the same epitope can be obtained by sequential panning. This method can be used to predict the location of some epitopes, but additional methods are needed to confirm the result.