bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurement of Different Forms of Tissue Plasminogen Activator in Plasma
Authors: Chandler Wayne L , Jascur Marcy L , Henderson Paul J 
Published: January 2020

Abstract
Background: We evaluated assays to measure both total tissue plasminogen activator (tPA) and the three principle forms of tPA in plasma: active tPA, tPA complexed with plasminogen activator inhibitor type 1 (PAI-1), and tPA complexed with C1-inhibitor.
Methods: Active tPA was measured by use of an indirect amidolytic assay and immunofunctional assays. tPA/PAI-1, tPA/C1-inhibitor, and total tPA antigen were measured by use of microtiter plates coated with anti-tPA antibodies and, respectively, anti-PAI-1, anti-C1-inhibitor, and anti-tPA antibodies conjugated to peroxidase.
Results: The immunofunctional tPA assay detected 1 U/L (0.001 U/mL) tPA and recovered 108% ± 12% of active tPA added to samples containing high (mean, 60 000 IU/L) PAI-1 activities vs a detection limit of 10 U/L (0.01 U/mL) and 13% ± 25% recovery for the indirect amidolytic tPA activity assay. For measurement of tPA/PAI-1 complex, polyclonal anti-PAI-1 conjugates recovered 112% ± 20% of the expected tPA/PAI-1 vs recovery of only 38% ± 16% when monoclonal anti-PAI-1 conjugates were used. Of three methods tested, two total tPA antigen assays correlated well (r2 = 0.85) and showed recoveries near 100%, whereas the third method showed lower correlations, higher intercepts, and falsely high recovery. A single anti-tPA capture antibody that performed the best in the individual assay evaluations was used to measure the different forms of tPA in 22 samples with a range of tPA and PAI-1 values. The sum of the molar concentrations of active tPA, tPA/PAI-1, and tPA/C1-inhibitor using the optimized methods was equal to 94% ± 7% of measured total tPA.
Conclusion: Optimized assays based on a single anti-tPA capture antibody can be used to accurately measure the major forms of tPA in plasma.
Simultaneous HPLC Assay for Quantification of Indinavir, Nelfinavir, Ritonavir, and Saquinavir in Human Plasma
Authors: Remmel Rory P , Kawle Sagar P , Weller Dennis , Fletcher Courtney V 
Published: January 2020

Abstract
Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed.
Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 μL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 × 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma.
Results: The extraction recovery was 87–92%. Within-run accuracy for quality-control samples was 6–8%, with CVs of 2–8%. Limits of quantification were 40–50 μg/L for indinavir, nelfinavir, and ritonavir, and 20 μg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 &gt;0.94) between methods. Larger variations at concentrations &gt;4000 μg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined.
Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications.
Pitfalls in the Diagnosis of Patients with a Partial Dihydropyrimidine Dehydrogenase Deficiency
Authors: Van Kuilenburg André B P , Van Lenthe Henk , Tromp Annelies , Veltman Patricia C J , Van Gennip Albert H 
Published: January 2020

AbstractBackground: Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-fluorouracil. To identify patients suffering from complete or partial DPD deficiency and to identify pitfalls that can preclude the proper diagnosis of patients with partial DPD deficiency, a sensitive and accurate assay is necessary.Methods: The activity of DPD was measured using [4-14C]thymine followed by separation of substrate and products with reversed-phase HPLC with on-line detection of the radioactivity.Results: Complete baseline separation of radiolabeled thymine and all degradation products was achieved within 15 min. The detection limit for dihydrothymine was 0.4 pmol. In lymphocytes, the DPD activity deviated from linearity at low protein concentrations (&lt;0.2 g/L). Profoundly decreased activity of DPD was detected in the peripheral blood mononuclear cells (PBM cells) of two tumor patients when measured at low protein concentrations. Low DPD activity comparable to that observed in obligate heterozygotes was initially detected in PBM cells, containing substantial amounts of myeloid cells, from a patient suffering from 5-fluorouracil toxicity. However, after the patient experienced full clinical recovery, normal DPD activity was observed in the PBM cells. No significant differences in DPD activity were observed between exponentially growing fibroblasts and those at confluence. The range of DPD activities of obligate heterozygotes overlaps the range of DPD activities of controls.Conclusions: The low activity of DPD measured in PBM cells containing myeloid cells or that measured at a low protein concentration in the assay mixture is not indicative of heterozygosity for a mutant DPD allele. Although fibroblasts are suitable to establish a complete deficiency of DPD, unambiguous detection of heterozygotes is not possible.
Enzymatic Mutation Detection in the P53 Gene
Authors: Inganäs Mats , Byding Sara , Eckersten Ann , Eriksson Solveig , Hultman Tomas , Jorsback Anneli , Löfman Esfir , Sabounchi Fariba , Kressner Ulf , Lindmark Gudrun , Tooke Nigel 
Published: January 2020

Abstract
Background: The enzymatic mutation detection (EMD) assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves preformed heteroduplex molecules at mismatch sites, forming two shorter fragments that can be resolved by gel electrophoresis. The method can be used to detect single and multiple base changes, as well as insertions and deletions.
Methods: The sensitivity, specificity, and positional accuracy of mutation detection by EMD with the PASSPORTTM Mutation Scanning Kit were assessed in a blind fashion for three analytical platforms (radioactive detection and automated laser sequencers ALFexpress and ABI PRISM 377). PCR products of 703 bp covering codons 188–393 of the P53 gene were prepared from colorectal tumor samples and analyzed by EMD; the results were compared to data from cDNA sequencing. A 1362-bp PCR product prepared from IL4r gene was used to test detection of multiple base changes in long PCR products.
Results: The sensitivity for detection of mutations using EMD exceeded 90%, and the specificity exceeded 80% on all analysis platforms. The method localized 90% of mutations to within two codons and four codons for automated laser sequencers and detection by radioactivity, respectively. The method detected at least five mismatches in heteroduplexes &gt;1 kb.
Conclusions: The EMD system facilitates efficient detection of genetic variation in fragments exceeding 1 kb irrespective of location and type. The technology is particularly well suited to the detection of mutations in genes frequently mutated at unpredictable locations.
Production and Characterization of Novel Anti-Prostate-specific Antigen (PSA) Monoclonal Antibodies That Do Not Detect Internally Cleaved Lys145-Lys146 Inactive PSA
Authors: Nurmikko Pauliina , Väisänen Ville , Piironen Timo , Lindgren Sari , Lilja Hans , Pettersson Kim 
Published: January 2020

AbstractBackground: The nature of free, uncomplexed prostate-specific antigen (PSA) in the circulation is still unknown. In this study, we developed novel anti-PSA antibodies using PSA produced by a metastasized cancer cell line, LNCaP, as an immunogen.Methods: Hybridoma cell lines were screened with different methods that aimed at finding antibodies specific for the forms of free PSA produced by LNCaP cell line. Obtained antibodies were further studied for their characteristics related to previously characterized monoclonal antibodies.Results: Numerous anti-PSA antibodies were obtained, of which four represented unique epitopes previously unrecognized by us. One free-PSA-specific antibody was bound to PSA on two distinct epitopes, and one antibody was bound to the carboxyl-terminal peptide of PSA. Two antibodies were found to bind to the peptide sequence adjacent to the internal cleavage site Lys145-Lys146. These antibodies failed to recognize internally cleaved PSA at Lys145-Lys146. We could not find anti-proPSA antibodies despite the fact that LNCaP PSA contained more than one-half of the zymogen form of PSA.Conclusions: We report, for the first time, novel anti-PSA antibodies that do not recognize internally cleaved PSA at Lys145-Lys146 and thus are specific for intact, unclipped PSA.
Testing Claimed Minimal Detectable Concentrations of in Vitro Medical Diagnostic Devices
Authors: Davenport James M , Schlain Brian 
Published: January 2020

Abstract
Manufacturers and users of medical diagnostic devices are provided a statistical decision tool for investigating a claimed minimal detectable concentration (MDC). The MDC is defined by setting two respective probabilities: that the blank sample being analyzed is determined to have analyte and that the device fails to determine a low concentration of analyte at the MDC. The statistical procedure for simultaneously testing the two aforementioned analytical decision errors assumes that signal responses follow a gaussian distribution but does not require a fitted calibration curve, knowledge of distribution parameters, or the assumption of constant variance in the low assay range. Evaluation of the operating characteristics of the procedure requires knowledge only of the variance ratio between the MDC and zero-dose signal distributions, which usually is well known.
Human Urine Certified Reference Material for Arsenic Speciation
Authors: Yoshinaga Jun , Chatterjee Amit , Shibata Yasuyuki , Morita Masatoshi , Edmonds John S 
Published: January 2020

Abstract
Background: Chemical speciation analysis is essential for the biological monitoring of inorganic arsenic exposure using urine as indicator medium. There is increasing demand for a certified reference material (CRM) of urine matrix for arsenic speciation.
Methods: Urine (10 L) was collected from non-occupationally exposed Japanese males. We prepared 954 bottles of urine, each containing ∼10 mL, after filtering and blending the urine stock. The urine in each bottle was freeze-dried. Between-bottle homogeneity was confirmed by measuring the concentrations of selected minor and trace elements in the material and subsequent statistical analysis. Certification was based on a collaborative analysis involving 15 laboratories.
Results: Certified values were determined for arsenobetaine (0.069 ± 0.012 mg As/L), dimethylarsinic acid (0.036 ± 0.009 mg As/L), and total arsenic (0.134 ± 0.011 mg/L) as well as for total selenium (0.059 ± 0.005 mg/L) and zinc (0.62 ± 0.05 mg/L), based on the analytical values from the collaborating laboratories. Reference values are given for copper (0.010 mg/L) and lead (0.0011 mg/L), based on definitive analysis at the National Institute for Environmental Studies (NIES).
Conclusions: The present CRM, NIES CRM No. 18 Human Urine, is the first human urine CRM for arsenic speciation and will be of value for analytical quality assurance of the biological monitoring of arsenic exposure.
Stability of Methylecgonidine and Ecgonidine in Sheep Plasma in Vitro
Authors: Scheidweiler Karl B , Shojaie Jalil , Plessinger Mark A , Wood Ronald W , Kwong Tai C 
Published: January 2020

AbstractBackground: Crack smokers are exposed to a pyrolysis product, methylecgonidine (MEG), which can be used as an analytical marker for crack smoking. Ecgonidine (EC), a hydrolytic product of MEG, has been identified in urine of crack smokers. MEG undergoes conversion to EC, complicating analysis and perhaps explaining a lack of forensic blood specimens containing MEG.Methods: We developed gas chromatography–mass spectrometry (GC-MS) assays for MEG and EC. Plasma was collected from sheep blood containing 0, 0.06, or 0.24 mol/L (0%, 0.25%, or 1%) NaF. MEG was added to these plasmas, and they were incubated at −80, 1, 21, or 37 °C to determine whether there were temporal, temperature, or storage effects on MEG stability over 48 h.Results: Decreased temperature and increased NaF concentrations limited MEG degradation and EC formation. MEG stored in plasma at −80 °C was stable up to 1 month, even in the absence of NaF.Conclusions: MEG is stable in sheep plasma collected in commercially available, evacuated blood-collection tubes containing NaF and stored at −80 °C. In vitro formation of EC can be minimized with appropriate sample handling, and its in vivo formation may provide a better marker of crack smoking than its parent pyrolysis product.
DNA Base Bulge vs Unmatched End Formation in Probe-based Diagnostic Insertion/Deletion Genotyping: Genotyping the UGT1A1 (TA)n Polymorphism by Real-Time Fluorescence PCR
Authors: von Ahsen Nicolas , Oellerich Michael , Schütz Ekkehard 
Published: January 2020

Abstract
Background: Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)nTAA insertion polymorphism (UGT1A1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1.17; UGT1A1). Present methods for genotyping this polymorphism are laborious.
Methods: Hybridization probes were designed complementary to the wild type (TA)6 and to alleles with (TA)7 and (TA)8 repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)5 to (TA)8 repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis.
Results: Only short probes (22–24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)7 or (TA)8 genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)7 allele.
Conclusions: The presented method for genotyping the (TA)n promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)n repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms.
Sensitive and Specific Immunodetection of Human Glandular Kallikrein 2 in Serum
Authors: Becker Charlotte , Piironen Timo , Kiviniemi Johanna , Lilja Hans , Pettersson Kim 
Published: January 2020

AbstractBackground: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2.Methods: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA.Results: The analytical detection limit was &lt;10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was &lt;0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4.8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-α1-antichymotrypsin (ACT) were detected in vitro with −6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4–19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor.Conclusions: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.
Sodium Pump Isoforms in Xenotransplantation: Importance of Biochemical Compatibility
Authors: Rose Andrea M , Qazzaz Hassan M , Zolotarjova Nina , Mellett Brenda J , Martin Alvin W , Valdes Jr Roland 
Published: January 2020

AbstractBackground: Xenotransplantation of pig hearts to humans could be hampered by the reportedly reduced affinity for digoxin of pig heart. We examined the hypothesis that expression of the individual α-subunit isoforms of the sodium pump [Na+,K+-ATPase (NKA)], the receptor for the plant-derived cardiac glycosides, may be responsible for this difference.Methods: We used a NKA-inhibition assay in combination with Western analysis, immunohistochemistry, and phosphorylation of the NKA α subunit to identify the distribution and expression of α isoforms in four chambers of porcine and human hearts.Results: We confirmed that tissue from porcine heart is less sensitive to digitalis (IC50 = 1740 nmol/L) when compared with human heart (IC50 = 840 nmol/L), whereas porcine cerebral cortex-mix had an affinity comparable to that of human heart (IC50 = 910 nmol/L). Our data show that porcine cerebral cortex-mix and human heart contain all three α isoforms, whereas porcine heart expresses only the α1 isoform.Conclusions: The different expressions of sodium pump isoforms in human vs porcine cardiac tissues suggests that porcine hearts may not be pharmacologically or endocrinologically compatible when used in humans. Studies of both pharmacologic and endocrinologic tissue compatibility are needed prior to selection of organs for xenotransplantation.
Development of a New Assay for Complex I of the Respiratory Chain
Authors: Brooks Hilary , Krähenbühl Stephan 
Published: January 2020

AbstractBackground: Measurement of complex I activity has been hampered by the large amounts of tissue required and the resulting turbidity of the assay solution, which makes spectrophotometric analysis difficult. We have developed a new assay for measuring the activity of complex I in isolated mitochondria that is also applicable to skeletal muscle homogenate in patients with suspected mitochondrial diseases.Methods: The method was a radioenzymatic assay based on the preferential oxidation of the 4B hydrogen of NADH by complex I. We prepared tritiated isoforms of NADH for both the respective 4A-3H and 4B-3H positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product (3H2O) was collected after charcoal adsorption of unreacted NADH and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity.Results: The assay was linear with time and protein for isolated mitochondria and tissue homogenates from rats and humans. The Vmax and Km values obtained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 μmol/L and 90 μmol · min−1 · mg protein−1, respectively. The assay was reproducible and useable for routine measurements in human skeletal muscle. The sensitivity was &gt;10-fold higher than the sensitivities of spectrophotometric techniques.Conclusions: The results of our studies demonstrate the successful development of a new assay for complex I that is rapid, easy to perform, and that enables the processing of multiple samples at one time.
Estimating the Long-Term Effects of Storage at −70 °C on Cholesterol, Triglyceride, and HDL-Cholesterol Measurements in Stored Sera
Authors: Shih Weichung Joe , Bachorik Paul S , Haga Jo A , Myers Gary L , Stein Evan A 
Published: January 2020

AbstractWe estimated the effects of long-term storage at −70 °C on serum total cholesterol, HDL-cholesterol, and triglycerides in specimens that had been stored for up to 7 years. These estimates were made using measurements in serial specimens collected from the placebo control group of the Air Force/Texas Coronary Atherosclerosis Prevention Study over a period of ∼5 years. We compared the group means for pairs of serial specimens taken at 6- and 12-month intervals, assuming that (a) a negligible placebo effect occurred between the serial specimen pairs; (b) in the absence of storage effects, the variation in the group means would reflect only normal biological variation and would not materially affect the group means for the serial specimens; (c) any systematic changes in these group means would reflect storage-related changes; and (d) storage-related changes are cumulative, i.e., the overall changes for a given storage period are the sum of the changes during previous storage periods. We observed average decreases of 2.0% per year for total cholesterol over 7 years and 2.8% per year in triglycerides for the first 5 years. HDL-cholesterol decreased by 1.3% per year, but this change was not statistically significant. This approach may be useful for estimating storage-related changes for studies in specimens stored for a period of years and for which stability data may not be available.