bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modified pentamer formation assay for measurement of tacrolimus and its active metabolites: comparison with liquid chromatography–tandem mass spectrometry and microparticle enzyme-linked immunoassay (MEIA-II)
Authors: Armstrong Victor W , Schuetz Ekkehard , Zhang Qingling , Groothuisen Stephan , Scholz Christa , Shipkova Maria , Aboleneen Hoda , Oellerich Michael 
Published: January 2020

Abstract
A modified pentamer formation assay (PFA) for quantification of tacrolimus and active metabolites after extraction from whole blood is described. The lower limit of detection was 2 μg/L. Intraassay precision (CV) was 5.7–13.7%, and the interassay CV was 6.1–14.9%. Tacrolimus trough concentrations in 104 whole blood specimens from liver and kidney transplant recipients were compared with results from HPLC–tandem mass spectrometry (LC/MS/MS) and microparticle enzyme immunoassay (MEIA-II). Data were analyzed by difference plots and are presented as median (95% confidence intervals) of the method differences. MEIA-II results were on average 2.00 μg/L (range, −0.08 to 5.17 μg/L) higher than LC/MS/MS, whereas PFA results were only 1.07 μg/L (range, −2.62 to 5.33 μg/L) higher. Of 104 specimens tested, 25 displayed differences ≥3 μg/L between MEIA-II and PFA: median difference, 4.65 μg/L (range, 3.01–8.79 μg/L). The corresponding median difference between PFA and LC/MS/MS was −0.91 μg/L (range, −4.11 to 0.85 μg/L), and the difference between MEIA-II and LC/MS/MS was 3.67 μg/L (range, 1.88–6.34 μg/L), suggesting the presence of inactive metabolites that caused a positive bias in the immunoassay. In contrast, similar median differences were observed for the remaining 79 specimens: MEIA-II minus LC/MS/MS, 1.78 μg/L (range, −0.45 to 4.11 μg/L); PFA minus LC/MS/MS, 1.90 μg/L (range, −1.70 to 5.50 μg/L). Active tacrolimus metabolites may have contributed to the higher apparent tacrolimus concentrations in these specimens.
When to collect blood specimens: midmorning vs fasting samples
Authors: Leppänen Esa , Dugué Benoit 
Published: January 2020

Abstract
This study was carried out to define the consequences of collecting blood specimens during the forenoon instead of using fasting specimens collected early in the morning. Extensive laboratory data were obtained from specimens collected from fasting participants at 0800, after breakfast at 0930, and again at 1100. The subjects were inpatients in medical and surgical wards (n = 51; 13 women and 38 men; ages, 32–87 years) and subjectively healthy volunteers corresponding to outpatients (n = 51; 31 women and 20 men; ages, 18–63 years). The coefficient of variation (CV, %) of the patient results was compared with the analytical CV. The observed CVs of the subjects’ results far exceeded the analytical CV (%), the average being 3.5-fold and up to 14-fold for some analytes. In individual results the observed change often exceeded the medically derived clinical critical difference. Laboratory data should always be interpreted in the context in which they were obtained. Clinical decisions should be based on objective data (observations) more than on experience and educated guesses. Different medical specialities and different clinical situations may require different kinds of procedures.
Improved method for measuring tartrate-resistant acid phosphatase activity in serum
Authors: Nakanishi Mamoru , Yoh Kousei , Uchida Kagehiro , Maruo Souji , Matsuoka Akira 
Published: January 2020

AbstractWe describe an improved method for the kinetic measurement of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. Of the TrACP derived from erythrocytes, platelets, and macrophages (osteoclasts and others), that from the first two sources is also resistant to fluoride, whereas skeletal TrACP is sensitive to fluoride. Thus, osteoclast-derived TrACP can be measured specifically by exploiting its sensitivity to fluoride. We measured the activity of tartrate-resistant and fluoride-sensitive acid phosphatase (TrFsACP) by using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2. The activity of TrFsACP in serum was increased by adding hexadimethrine bromide (Polybrene) to the reaction mixture. This method was not influenced by hemolysis with hemoglobin concentrations as great as 0.9 g/L. The mean ± SD values of TrFsACP activity by this method were 20.4 ± 2.8 and 16.4 ± 2.3 U/L for young (ages 20–29 years) men (n = 34) and women (n = 50), respectively. The highest mean TrFsACP activity was found among children younger than 15 years, followed by that in elderly subjects (older than 60).
Transient 5-oxoprolinuria (pyroglutamic aciduria) with systemic acidosis in an adult receiving antibiotic therapy
Authors: Croal Bernard L , Glen Alastair C A , Kelly Christopher J G , Logan Robert W 
Published: January 2020

Abstract
5-Oxoprolinuria is a recognized condition with increased urinary excretion of 5-oxoproline and is associated with a variety of inborn metabolic defects involving the series of enzyme-linked reactions known as the γ-glutamyl cycle. We report the unusual case of a 35-year-old woman who initially presented with staphylococcal pneumonia but went on to develop a transient high anion gap metabolic acidosis. The development and subsequent complete recovery from this acidosis were subsequently shown to be related in time to the intravenous administration of the antibiotics flucloxacillin and netilmicin. Analysis of the patient’s urine for organic acids revealed massively increased excretions of 5-oxoproline at the peak of her acidosis. We suggest that this patient developed a transient disturbance in the γ-glutamyl cycle involving the 5-oxoprolinase step, which resulted in accumulation of 5-oxoproline that caused a severe high anion gap metabolic acidosis. The administered antibiotics remain as possible causative agents.
Supporting pharmaceutical studies for FDA submissions: diversifying the drug monitoring laboratory
Authors: Moyer Thomas P , Oliver Lawrence K 
Published: January 2020

Abstract
Well-founded pharmacokinetic information is one of the cornerstones of a New Drug Application (NDA) to the Food and Drug Administration (FDA) required to introduce a new drug or a generic equivalent (ANDA) to the marketplace. The service that laboratories engaged in therapeutic drug monitoring provide to support clinical activities is also needed by the pharmaceutical industry during the evaluation and introduction of drugs to the marketplace. In considering this alternative service activity, one must be aware of and compliant with rules established by the FDA for performance of such studies. As specified in CFR 21, Parts 58, 211, and 320, good clinical and laboratory practice indicates that the laboratory should employ a Lab Study Director, who is responsible for the validation of all procedures implemented to support a study protocol, ensures that the laboratory carries out the study following these defined procedures, and personally reviews the results of all testing. The laboratory must validate each procedure by demonstrating and documenting that the procedure does what it is designed to do while meeting the analytical performance specifications required by the study. Laboratory records of all activities must be maintained and available for inspection by the FDA on request. The FDA has authority over all activities related to NDA and ANDA submissions and can bring criminal charges if results of a study are changed because a laboratory deviates from standard procedure. Competent drug monitoring laboratories are fully capable of participating in clinical trials testing activities. Laboratory staff should be fully versed in the FDA rules governing these activities, validate all procedures, and establish systems to verify the procedures are carried out as specified.
Simultaneous analysis of plasma free fatty acids and their 3-hydroxy analogs in fatty acid β-oxidation disorders
Authors: Costa Catarina G , Dorland Lambertus , Holwerda Ulbe , Tavares de Almeida Isabel , Poll-The Bwee-Tien , Jakobs Cornelis , Duran Marinus 
Published: January 2020

AbstractWe present a new derivatization procedure for the simultaneous gas chromatographic–mass spectrometric analysis of free fatty acids and 3-hydroxyfatty acids in plasma. Derivatization of target compounds involved trifluoroacetylation of hydroxyl groups and tert-butyldimethylsilylation of the carboxyl groups. This new derivatization procedure had the advantage of allowing the complete baseline separation of free fatty acids and 3-hydroxyfatty acids while the superior gas chromatographic and mass spectrometric properties of tert-butyldimethylsilyl derivatives remained unchanged, permitting a sensitive analysis of the target compounds. Thirty-nine plasma samples from control subjects and patients with known defects of mitochondrial fatty acid β-oxidation were analyzed. A characteristic increase of long-chain 3-hydroxyfatty acids was observed for all of the long-chain 3-hydroxyacyl-CoA dehydrogenase-deficient and mitochondrial trifunctional protein-deficient plasma samples. For medium-chain acyl-CoA dehydrogenase deficiency and very-long-chain acyl-CoA dehydrogenase deficiency, decenoic and tetradecenoic acids, respectively, were the main abnormal fatty acids, whereas the multiple acyl-CoA dehydrogenase-deficient patients showed variable increases of these unusual intermediates. The results showed that this selective and sensitive method is a powerful tool in the diagnosis and monitoring of mitochondrial fatty acid β-oxidation disorders.
Expression of prostate-specific antigen and prostate-specific membrane antigen transcripts in blood cells: implications for the detection of hematogenous prostate cells and standardization
Authors: Gala Jean-Luc , Heusterspreute Michel , Loric Sylvain , Hanon France , Tombal Bertrand , Van Cangh Paul , Philippe De Nayer  , Philippe Marianne 
Published: January 2020

Abstract
Circulating prostate cells can be detected in cancer patients by using reverse transcriptase–PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.
Molecular beacons: a new approach for semiautomated mutation analysis
Authors: Giesendorf Belinda A J , Vet Jacqueline A M , Tyagi Sanjay , Mensink Ewald J M G , Trijbels Frans J M , Blom Henk J 
Published: January 2020

Abstract
Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We designed molecular beacons to detect a point mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, a mutation that has been related to an increased risk for cardiovascular disease and neural tube defects. The application of molecular beacons enables fast, semi- automated, accurate mutation detection. Moreover, the procedure is performed in a closed tube system, thereby avoiding carryover contamination. We believe these probes will find their way into nucleic acid research and diagnostics.
Human cardiac troponin I: precise identification of antigenic epitopes and prediction of secondary structure
Authors: Ferrieres Gaelle , Calzolari Charles , Mani Jean-Claude , Laune Daniel , Trinquier Sylvie , Laprade Michel , Larue Catherine , Pau Bernard , Granier Claude 
Published: January 2020

AbstractThe presence of human cardiac troponin I (hcTnI) in serum is considered to be a highly specific biochemical marker of acute myocardial infarction. To better understand the antigenic properties of hcTnI, a set of 68 overlapping peptides covering the complete amino acid sequence of hcTnI was prepared and used in epitope mapping experiments. All 16 anti-hcTnI monoclonal antibodies tested were found to recognize a peptide epitope, indicating that recognition by anti-hcTnI monoclonal antibodies was not dependent on the tertiary structure of the protein. Furthermore, the peptide reactivity with anti-hcTnI polyclonal antibodies indicated that most of the sequence of the protein was antigenic; in particular, the N- and C-terminal extremities were found to be the strongest antigenic regions. By using accurate secondary structure prediction methods, hcTnI was found to be an all-alpha type protein, with five regions predicted as helices. Matching the results of the epitope analysis with the structural prediction led us to the view that hcTnI is not a globular protein but probably adopts an extended conformation, allowing a large part of the amino acid sequence of this molecule to be recognized by the immune system. This improved knowledge of the antigenic and structural properties of hcTnI may help in developing new antibodies and immunoassays for use in diagnosing myocardial infarction.
Chondrex: new marker of joint disease
Authors: Harvey Sheryl , Weisman Michael , O’Dell James , Scott Tonya , Visor Mindy Krusemeier Jill , Swindlehurst Cathy 
Published: January 2020

AbstractChondrex, a major secretory protein of human chondrocytes and synovial fibroblasts, is increased in serum of patients with joint and cartilage disease. We have developed a sandwich-type ELISA for quantifying chondrex in serum. The interassay CVs were 2.8–3.7% and the average within-run and total CVs were 3.6% and 5.4%, respectively. The limit of detectability by linear dilution was 20 μg/L, recovery upon dilution was 102% ± 5%, and analytical recovery (of added analyte) was 98% ± 11%. The reference interval (central 90% interval) for chondrex in healthy adults was 25–95 μg/L. Chondrex values for patients with active rheumatoid arthritis or osteoarthritis were significantly greater than in healthy adults, inactive rheumatoid arthritis patients, and diabetes patients (P &lt;0.05). In patients treated with disease-modifying antirheumatic drug therapy, decreasing chondrex values reflected the clinical improvement observed in responders, whereas the values were maintained or increased in nonresponders. In conclusion, chondrex may be a useful marker in the clinical investigation of arthritis.
Effect of arsenosugar ingestion on urinary arsenic speciation
Authors: Ma Mingsheng , Le X Chris 
Published: January 2020

Abstract
We developed and evaluated a method for the determination of μg/L concentrations of individual arsenic species in urine samples. We have mainly studied arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) because these are the most commonly used biomarkers of exposure by the general population to inorganic arsenic and because of concerns over these arsenic species on their toxicity and carcinogenicity. We have also detected five unidentified urinary arsenic species resulting from the metabolism of arsenosugars. We combined ion pair liquid chromatography with on-line hydride generation and subsequent atomic fluorescence detection (HPLC/HGAFS). Detection limits, determined as three times the standard deviation of the baseline noise, are 0.8, 1.2, 0.7, and 1.0 μ/L arsenic for arsenite, arsenate, MMAA, and DMAA, respectively. These correspond to 16, 24, 14, and 20 pg of arsenic, respectively, for a 20-μL sample injected for analysis. The excellent detection limit enabled us to determine trace concentrations of arsenic species in urine samples from healthy subjects who did not have excess exposure to arsenic. There was no need for any sample pretreatment step. We used Standard Reference Materials, containing both normal and increased concentrations of arsenic, to validate the method. Interlaboratory studies with independent techniques also confirmed the results obtained with the HPLC/HGAFS method. We demonstrated an application of the method to the determination of arsenic species in urine samples after the ingestion of seaweed by four volunteers. We observed substantial increases of DMAA concentrations in the samples collected from the volunteers after the consumption of seaweed. The increase of urinary DMAA concentration is due to the metabolism of arsenosugars that are present in the seaweed. Our results suggest that the commonly used biomarkers of exposure to inorganic arsenic, based on the measurement of arsenite, arsenate, MMAA, and DMAA, are not reliable when arsenosugars are ingested from the diet.
Radioimmunoassay of rat leptin: sexual dimorphism reversed from humans
Authors: Landt Michael , Gingerich Ronald L , Havel Peter J , Mueller Wendy M , Schoner Bridgette , Hale John E , Heiman Mark L 
Published: January 2020

AbstractAdipose tissue secretes leptin, which interacts with receptors in the hypothalamus. In rodent models of obesity, leptin increases metabolism and decreases food intake, which helps to maintain normal body composition. Accurate and precise methods to quantitate circulating leptin concentrations are needed for physiological studies. We developed an RIA to measure leptin in rat plasma, serum, or adipocyte culture fluids. The working range of the assay, defined by the detection limit and the highest calibrator, was 0.5–50 μg/L. Recovery of 1.6–11.6 μg/L leptin added to serum was 92–103%. The rat leptin RIA correlated well with a previously developed mouse RIA when rat plasma was assayed with both methods (r = 0.94), but the mouse leptin assay underestimated rat leptin in plasma. Within- and between-run CVs were 2.4% to 5.7%. Plasma leptin concentrations correlated directly with percentage of body fat, and correlation improved when the results were separated by gender (r = 0.796, P &lt;0.001 for males; r = 0.710, P &lt;0.001 for females). Leptin concentrations were generally higher in male rats than in females; plasma leptin increased 0.60 μg/L for each percentage of increase in body fat for males but only 0.22 μg/L for females. We conclude that rat serum/plasma leptin concentrations are accurately and precisely measured with this new RIA.
Multicenter evaluation of the Paragon CZETM 2000 capillary zone electrophoresis system for serum protein electrophoresis and monoclonal component typing
Authors: Bienvenu Jacques , Graziani Maria Stella , Arpin François , Bernon Hélène , Blessum Cynthia , Marchetti Cristina , Righetti Gabriella , Somenzini Manuela , Verga Gioacchino , Aguzzi Francesco 
Published: January 2020

Abstract
Serum protein electrophoresis and typing of monoclonal components (MCs) are routine but time-consuming and technically demanding assays. We evaluated capillary electrophoresis (Paragon CZETM 2000) for automation of the two assays. CZE and cellulose acetate electrophoresis gave similar data on 794 samples. Within-run and between-run CVs were &lt;2% for albumin and γ-globulins and 4–7% for α1-, α2-, and β-globulins. Bilirubin, hemoglobin, triglycerides, and fibrinogen were found not to interfere. No carryover by capillaries was detected. The detection limit for MC was &lt;0.5 g/L. MC assessment by immunosubtraction on 403 samples identified the monoclonal type in all samples with peak concentrations &gt;10 g/L; only 50% of MCs that could not be quantified by densitometric scan were typed.
Improved protamine-sensitive membrane electrode for monitoring heparin concentrations in whole blood via protamine titration§
Authors: Ramamurthy Narayanan , Baliga Narayan , Wahr Joyce A , Schaller Ulrich , Yang Victor C , Meyerhoff Mark E 
Published: January 2020

AbstractAn improved protamine-sensitive electrode based on a polymeric membrane doped with the charged ion exchanger dinonylnaphthalenesulfonate (DNNS) is used for monitoring heparin concentrations in whole blood. The electrode exhibits significant nonequilibrium potentiometric response to polycationic protamine over the concentration range of 0.5–20 mg/L in undiluted whole-blood samples. The sensor can serve as a simple end point detector for the determination of heparin via potentiometric titrations with protamine. Whole-blood heparin concentrations determined by the electrode method (n ≥157) correlate well with other protamine titration-based methods, including the commercial Hepcon HMS assay (r = 0.934) and a previously reported potentiometric heparin sensor-based method (r = 0.973). Reasonable correlation was also found with a commercial chromogenic anti-Xa heparin assay (r = 0.891) with corresponding plasma samples and appropriate correction for whole-blood hematocrit levels. Whereas a significant positive bias (0.62 kU/L; P &lt;0.001) is observed between the anti-Xa assay and the protamine sensor methods, insignificant bias is observed between the protamine sensor and the Hepcon HMS tests (0.08 kU/L; P = 0.02). The possibility of fully automating these titrations offers a potentially simple, inexpensive, and accurate method for monitoring heparin concentrations in whole blood.
Multicenter harmonization of common enzyme results by fresh patient-pool sera
Authors: Franck Paul F H , Steen Gerard , Lombarts Arnold J P F , Souverijn John H M , van Wermeskerken Robert K A 
Published: January 2020

Abstract
A region consisting of 19 clinical laboratories harmonized their calibration of seven common enzymes by using fresh patient-pool sera. One of the laboratories was chosen to act as Regional Reference Laboratory (RRL). This laboratory used internationally accepted (mostly IFCC) methods at 37 °C, with an intralaboratory CV ≤2.5%. First, the reference ranges of the RRL were verified by analysis of a reference population and calculation of the results by a parametric method. Next, all laboratories, including the RRL, received six patient-pool sera and analyzed them at the same time on the same date. Enzyme calibration factors at each laboratory were converted on the basis of the slope, and occasionally the intercept, of regression analysis with the RRL and the individual laboratory. Before harmonization, the interlaboratory CVs varied from 16.9% to 61.6%. After harmonization, CVs decreased to between 5.0% and 9.5%. These results proved to be reproducible over a period of more than a year. Using internationally accepted inaccuracy and imprecision criteria, the achieved interlaboratory CVs permit the use of one set of reference ranges by all participating laboratories. Certified Reference Materials were analyzed, resulting in interlaboratory CVs as low as those achieved with patient-pool sera. These materials can act as commutable reference preparations, except for creatine kinase.