bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clinical Significance of Immunoassays for Type-5 Tartrate-resistant Acid Phosphatase
Authors: Nakasato Yuri R , Janckila Anthony J , Halleen Jussi M , Vaananen H Kalervo , Walton Stephanie P , Yam Lung T 
Published: January 2020

AbstractBackground: Tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) is a product of osteoclasts and a biochemical marker of bone resorption rate. However, erythrocytes and platelets contribute to total TRAP activity in serum, reducing the specificity of direct biochemical assays in serum. Osteoclast TRAP is also known as type-5 TRAP and is antigenically unique. Immunoassays are sought to improve the specificity and sensitivity of TRAP as a bone marker.Methods: We developed two colorimetric microplate assays for type-5 TRAP: an enzyme capture immunoassay to measure antibody-bound enzymatic activity, and a two-site immunoassay to measure bound enzyme protein. Both use the same monoclonal antibody (14G6) to capture type-5 TRAP, which permits determination of specific activity of serum TRAP in health and disease.Results: Both TRAP assays were linear from one-tenth to fivefold the mean value in 18 healthy subjects. In these subjects, the mean (SD) TRAP activity was 3.2 (0.54) U/L for the enzyme capture assay and 37 (13) μg/L for the two-site assay. Mean TRAP activity was not significantly increased in 64 patients with endstage renal disease requiring hemodialysis (HD) or 99 unselected patients with rheumatic diseases. By contrast, TRAP protein was increased in both the HD and rheumatic disease groups. The specific activity of TRAP in the 17 of 64 HD sera that had increased TRAP activity (0.088 U/μg) was similar to that in healthy subjects (0.091 U/μg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/μg) was significantly decreased.Conclusions: Wide sample distributions for TRAP activity in HD patients and TRAP protein in rheumatic disease patients suggest the presence of subpopulations of HD patients with increased TRAP activity and of rheumatic patients with increased TRAP protein. Each assay for TRAP activity and protein may have its own biological significance and clinical applications in specific groups of patients.
Efficient Cleavage of Conjugates of Drugs or Poisons by Immobilized β-Glucuronidase and Arylsulfatase in Columns
Authors: Toennes Stefan W , Maurer Hans H 
Published: January 2020

AbstractBackground: Cleavage of conjugates is an important step in toxicological analysis, especially of urine samples. The aim of this study was to combine the advantages and to reduce the disadvantages of acid hydrolysis and conventional enzymatic hydrolysis procedures.Methods: β-Glucuronidase (GRD; EC 3.2.1.31) and arylsulfatase (ARS; EC 3.1.6.1) were purified and coimmobilized on an agarose gel matrix and packed into columns.Results: In columns packed with GRD and ARS, the test conjugates 4-nitrophenyl glucuronide and 4-nitrophenyl sulfate added into urine could be completely cleaved within 25 min. Even the relatively stable morphine conjugates could be completely hydrolyzed within 60 min in authentic urine samples. Therefore, an incubation time of 1 h is recommended. Enzyme inhibition by matrix or by rather high concentrations of acetaminophen conjugates was tested and found to be up to 50%. However, a large excess of GRD and ARS was used. The immobilizate columns could be reused for at least 70 incubations and had a storage stability of at least 12 weeks. Carryover of analytes in reused columns could be avoided by rinsing with 200 mL/L methanol in acetate buffer. Thus, five drugs known to be contaminants added in very high concentrations into urine could be completely removed from the columns. A study on the applicability in systematic toxicological analysis showed that 120 different drugs and/or their metabolites could be detected in 35 different authentic urine samples.Conclusions: Use of immobilized and column-packed GRD and ARS is an efficient alternative for the cleavage of urinary conjugates in clinical toxicology.
Dihydrofolate Reductase Enzyme Inhibition Assay for Plasma Methotrexate Determination Using a 96-Well Microplate Reader
Authors: Widemann Brigitte C , Balis Frank M , Adamson Peter C 
Published: January 2020

Abstract
Microplate reader assays offer several advantages over conventional spectrophotometric assays. We adapted the dihydrofolate reductase (DHFR) enzyme inhibition assay for use in a 96-well microplate reader to measure plasma methotrexate (MTX) concentrations. The assay is linear from 0.01 to 0.1 μmol/L. The within-run CVs at 0.03 μmol/L and 0.08 μmol/L MTX were 4.0% and 2.7%, respectively, and the interday (total) CVs were 7.6% and 1.8%. Cross-reactivity with the inactive MTX metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA) was 3.9%, significantly less than that described with commercial immunoassays; with 7-hydroxymethotrexate cross-reactivity was 1.7%. In addition to sensitivity and specificity, the advantages of this assay are small sample volumes, simultaneous analysis of multiple samples, and rapid turnaround. Because of its greater specificity, the DHFR enzyme inhibition assay may be useful when DAMPA is present in plasma samples and HPLC is not available.
Influence of Aromatase Inhibitors on Plasma Total Homocysteine in Postmenopausal Breast Cancer Patients
Authors: Anker Gun B , Refsum Helga , Ueland Per M , Johannessen Dag C , Lien Ernst A , Lønning Per E 
Published: January 2020

AbstractIn this study, we evaluated the effect of estrogen suppression with three aromatase inhibitors, aminoglutethimide (n = 30), formestane (n = 12), and exemestane (n = 10), and the progestin megestrol acetate (n = 21) on plasma total homocysteine (tHcy) in patients suffering from advanced breast cancer. Treatment with 1 g/day aminoglutethimide for 2 and 3–5 months increased plasma tHcy by a mean value of 24.5% [95% confidence interval, 10.5–40.4%] at 2 months and 35.8% (95% confidence interval, 18.2–55.9%) at 3–5 months, corresponding to increases in the mean plasma tHcy of 1.90 and 3.67 μmol/L, respectively. In contrast, none of the other treatment options influenced plasma tHcy concentrations. The finding that aminoglutethimide, but none of the other aromatase inhibitors or megestrol acetate, influenced plasma tHcy suggests that this effect is achieved by mechanisms not related to suppression of plasma estrogens or to the glucocorticoids administered in concert.
Allele-specific Differences in Apolipoprotein C-III mRNA Expression in Human Liver
Authors: Esterbauer Harald , Hell Emanuel , Krempler Franz , Patsch Wolfgang 
Published: January 2020

AbstractBackground: Sequence variations at the apolipoprotein (apo)C-III gene locus have been associated with increased plasma triglycerides. In particular, the S2 allele of an SstI polymorphism in the 3′ untranslated region has been associated with hypertriglyceridemia in many populations. The aim of this study was to determine whether the variant S2 allele is related to increased mRNA expression in vivo.Methods: We measured allele-specific apoC-III expression in liver biopsies of five obese subjects, using restriction isotyping and a primer extension method, both based on the SstI polymorphism.Results: The expression of mRNA by the S1 and S2 alleles was similar in two patients, whereas the mRNA encoded by the S2 allele was 14%, 26%, and 29% more abundant than the wild-type mRNA in the remaining three patients. Because other polymorphisms at the apoC-III gene locus have been implicated in the S2-associated hypertriglyceridemia, we determined apoC-III haplotypes comprising promoter polymorphisms at −935, −641, −630, −625, −482, −455, as well as the SstI sites and a BbvI site, both located in the 3′ untranslated region. None of these polymorphisms nor any haplotype exhibited a perfect association with allele-specific expression, but variation at the T-482C site correlated in four of five subjects with the relative allele abundance.Conclusion: These data provide preliminary evidence for allele-specific differences in apoC-III mRNA expression in vivo and suggest that such differences may contribute to associations of apoC-III gene polymorphisms with hypertriglyceridemia.
Survey of Total Error of Precipitation and Homogeneous HDL-Cholesterol Methods and Simultaneous Evaluation of Lyophilized Saccharose-containing Candidate Reference Materials for HDL-Cholesterol
Authors: Cobbaert Christa , Mulder Paul GH , Baadenhuijsen Henk , Zwang Louwerens , Weykamp Cas W , Demacker Pierre NM 
Published: January 2020

Abstract
Background: Standardization of HDL-cholesterol is needed for risk assessment. We assessed for the first time the accuracy of HDL-cholesterol testing in The Netherlands and evaluated 11 candidate reference materials (CRMs).
Methods: The total error (TE) of HDL-cholesterol measurements was assessed in native human sera by 25 Dutch clinical chemistry laboratories. Concomitantly, the suitability of lyophilized, saccharose-containing CRMs (n = 11) for HDL-cholesterol was evaluated.
Results: In the precipitation method group, which included 25 laboratories and four methods, the mean (minimum–maximum) TE was 11.5% (2.7–25.2%), signifying that 18 of 25 laboratories satisfied the TE goal of ≤13% issued by the National Cholesterol Education Program (NCEP). In the homogeneous HDL-cholesterol method group, which included five laboratories, each performing two different methods, the mean (minimum–maximum) TE was 9.5% (6.0–17.3%) for the Boehringer assay and 15.7% (3.3–30.7%) for the Genzyme assay. For the Boehringer homogeneous assay, one of five laboratories did not meet the TE criterion, whereas for the Genzyme homogeneous assay, three of five laboratories exceeded the 13% criterion. The biases on the HDL-cholesterol values found by various precipitation methods were highly variable in all CRMs, irrespective of the quality, whereas the biases found by the homogeneous method from Boehringer were far less than ±5% for the highest-quality CRMs (CRMs 4–6).
Conclusions: The NCEP goal was met by 24 of 35 laboratories assessed by use of native human sera. Selectively pooled, lyophilized CRMs that are cryoprotected with 200 g/L saccharose have ample potential for use in the standardization of homogeneous HDL-cholesterol methods.
Assessment of Serum Thyroxine Binding Capacity-dependent Biases in Free Thyroxine Assays
Authors: Christofides Nicos D , Wilkinson Ewan , Stoddart Mary , Ray David C , Beckett Geoff J 
Published: January 2020

Abstract
Background: Free thyroxine (FT4) assays may exhibit biases that are related to serum T4 binding capacity (sBC). We describe two tests that can be used to assess the presence and magnitude of sBC-dependent biases in FT4 assays.
Methods: We used a direct equilibrium dialysis FT4 assay as the reference method and compared the results obtained with those of the FT4 assays under investigation, in patient sera having a wide range of sBC. We then compared the expected and observed FT4 results for sera diluted with an inert buffer. Because serum dilution causes a predictable decrease in sBC, an increasingly negative bias on progressive dilution is indicative of a sBC-dependent bias.
Results: The automated FT4 assay investigated (Vitros FT4) showed no demonstrable sBC-dependent bias by either test.
Conclusion: These two tests can be used to screen for sBC-dependent biases in FT4 assays.
Within- and Between-Subject Variation in Commonly Measured Anthropometric and Biochemical Variables
Authors: Widjaja Adji , Morris Richard J , Levy Jonathan C , Frayn Keith N , Manley Susan E , Turner Robert C 
Published: January 2020

Abstract
Background: The biological variation of some commonly assessed metabolic variables in healthy subjects has not been studied extensively. The aim of the study was to assess, in 12 healthy subjects (6 male and 6 female; mean (SD) age; 22.7 (1.5) years) following an overnight fast, the day-to-day variation of body fat (impedance method), triglycerides, nonesterified fatty acid (NEFAs), glycerol, 3-hydroxybutyrate (3-OHB), lactate, glucose, insulin (RIA), C-peptide, and glucagon on 12 consecutive days.
Methods: Between- and within-subject coefficients of variation (CVG and CVW) were estimated using a random effects analysis of variance, and assay variation was subtracted to give the coefficient of within-subject biological variation (CVI). Individuality indices were calculated as CVW/CVG.
Results: The overall means, CVI, and individuality indices were as follows: for body fat, 24.2%, 10%, and 0.3; for triglycerides, 0.61 mmol/L, 21%, and 1.1; for NEFAs, 376 μmol/L, 45%, and 1.4; for glycerol, 48 μmol/L, 36%, and 0.8; for 3-OHB, 43 μmol/L, 61%, and 1.5; for lactate, 0.88 mmol/L, 31%, and 1.1; for glucose, 4.9 mmol/L, 4.8%, and 0.7; for insulin, 52 pmol/L, 26%, and 1.0; for C-peptide, 0.39 nmol/L, 24%, and 0.9; and for glucagon, 53 ng/L, 19%, and 0.8.
Conclusions: The data presented here are necessary for the evaluation of several important metabolic variables in individual and group studies. The biological variation of some metabolites makes it difficult to characterize the status of healthy subjects with a single measurement.
Measurement of Plasma Renin Activity with Use of HPLC-Electrospray-Tandem Mass Spectrometry
Authors: Fredline Victoria F , Kovacs Eva M , Taylor Paul J , Johnson Anthony G 
Published: January 2020

AbstractBackground: The measurement of renin activity is complicated by difficulties in the quantification of angiotensin 1 (Ang1), the product of the renin-catalyzed reaction. We report an HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of Ang1 as a measure of plasma renin activity (PRA).Methods: After incubation (37 °C for 3 or 18 h), samples were prepared using C18 solid-phase extraction. [Val]5Ang1 was used as the internal standard (IS). Chromatography was performed on a C18 column, using 200 mL/L ammonium acetate buffer–800 mL/L methanol as the mobile phase. The flow rate was 150 μL/min, with a chromatographic run time of 5 min/sample. Mass spectrometric detection was in the positive ionization mode with selected reaction monitoring (Ang1 m/z 649.0→784.0; IS m/z 641.9→770.4).Results: The assay was linear over the range 2.5–500 ng Ang1/mL, which corresponded to a limit of detection (signal-to-noise ratio of 3:1) of PRA of 0.14 ng Ang1 · mL−1 · h−1. The imprecision (CV) of the assay at PRA values of 26.1, 13.5, 3.2, and 0.78 ng Ang1 · mL−1 · h−1 was 7.0%, 7.0%, 15%, and 11%, respectively. Absolute recoveries were 92.3% (Ang1) and 87.4% (IS). Incubation times of 3 h vs 18 h in the PRA assay gave good agreement at PRA &lt;2 ng Ang1 · mL−1 · h−1, but samples with a PRA of 2–5 ng Ang1 · mL−1 · h−1 gave lower PRA results after incubation for 18 h than after 3 h. We compared the HPLC-ESI-MS/MS assay and an RIA for the determination of PRA, with PRA incubation times of 3 h and 1.5 h, respectively. The mean PRA based on RIA of Ang1 was higher than that obtained using HPLC-ESI-MS/MS.Conclusion: The HPLC-ESI-MS/MS method allows sensitive and specific measurement of PRA. The higher activities measured with the RIA method highlight its potential for overestimation of PRA.
Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET
Authors: Little Michael C , Andrews Jeffrey , Moore Richard , Bustos Silvia , Jones Lynda , Embres Chris , Durmowicz Gerard , Harris James , Berger Dolores , Yanson Karen , Rostkowski Christine , Yursis Daretta , Price James , Fort Thomas , Walters Adriann , Collis Matthew , Llorin Oscar , Wood Janet , Failing Frank , O’Keefe Christian , Scrivens Brian , Pope Bill , Hansen Tim , Marino Ken , Williams Keith , Boenisch Michael 
Published: January 2020

Abstract
Background: Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations.
Methods: We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx® amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae.
Results: Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx.
Conclusions: The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C.trachomatis and N.gonorrhoeae and other organisms.
Assay of Procarboxypeptidase U, a Novel Determinant of the Fibrinolytic Cascade, in Human Plasma
Authors: Schatteman Katinka A , Goossens Filip J , Scharpé Simon S , Neels Hugo M , Hendriks Dirk F 
Published: January 2020

AbstractBackground: Procarboxypeptidase U (proCPU) is a novel proenzyme found in human plasma. The active form, carboxypeptidase U (CPU; EC 3.4.17.20), retards the rate of fibrinolysis through its ability to cleave C-terminal lysine residues on fibrin partially degraded by plasmin. This reduces the number of high-affinity plasminogen-binding sites on fibrin.Methods: We developed an assay to determine the proCPU concentration in human plasma. The assay involved quantitative conversion of proCPU to active CPU by thrombin-thrombomodulin, a very efficient activator of proCPU, followed by determination of the enzymatic activity of CPU with the substrate hippuryl-l-arginine, using an HPLC-assisted determination of the released hippuric acid. Using this method, we established a reference interval based on 490 healthy individuals.Results: The mean proCPU concentration, determined after activation of the zymogen in diluted plasma and expressed as CPU activity, was 964 U/L, with a SD of 155 U/L. The population showed a gaussian distribution. However, we noticed important differences related to age and the use of hormone preparations.Conclusions: The sensitivity and precision of the method make it suitable for routine clinical determinations and as a reference procedure.
Hyperinsulinemia and Related Atherosclerotic Risk Factors in the Population at Cardiovascular Risk: A Community-based Study
Authors: Chien Kuo-Liong , Lee Yuan-Teh , Sung Fung-Chang , Hsu Hsiu-Ching , Su Ta-Chen , Lin Ruey S 
Published: January 2020

AbstractBackground: A population-based study was conducted in Taiwan to investigate the prevalence of insulin resistance and high serum insulin concentrations and their relationships with potential atherosclerotic risk factors.Methods: We studied 2165 subjects, ages &gt;35, from a community cohort.Results: The distributions of fasting insulin were skewed to the right, with higher concentrations in women than in men. As age increased, insulin increased in women, but decreased in men. As fasting insulin concentrations increased, postloading insulin, glucose, blood pressure, body mass index, waist-to-hip ratio, total cholesterol, triglycerides, LDL-cholesterol, apoprotein B, plasminogen activator inhibitor 1, tissue plasminogen activator, and fibrinogen increased, but lipoprotein(a), HDL-cholesterol, and apoprotein A1 decreased. Multiple logistic regression showed that obesity, high LDL-cholesterol, and low HDL-cholesterol were significant predictors of hyperinsulinemic status.Conclusion: The study subjects with insulin resistance syndrome and related risk factors may be at risk for atherosclerosis, thrombosis, and other coronary heart diseases.
Use of Bone Alkaline Phosphatase to Monitor Alendronate Therapy in Individual Postmenopausal Osteoporotic Women
Authors: Kress Barry C , Mizrahi Isaac A , Armour Karen W , Marcus Robert , Emkey Ronald D , Santora Arthur C 
Published: January 2020

AbstractBackground: Biochemical bone markers are sensitive to the changes in bone turnover that result from treatment of postmenopausal osteoporotic women with antiresorptive therapies. Although information is available on the use of bone markers in monitoring therapy in groups of subjects, less is known regarding how these markers perform in individual patients.Methods: Serum bone alkaline phosphatase (bone ALP) concentrations, measured with the Tandem® Ostase® assay, were used to monitor the biochemical response of bone in postmenopausal women with osteoporosis receiving either 10 mg/day alendronate therapy (n = 74) or calcium supplementation (n = 148) for 24 months.Results: Bone ALP decreased significantly from baseline at 3 months (P ≤0.0001), reaching a nadir between 3 and 6 months of alendronate therapy. The magnitude of the bone ALP decrease in the treated osteoporotic population was consistent with normalization to premenopausal concentrations. Of the 74 alendronate-treated subjects, 63 (85.1%) demonstrated a decrease from baseline in bone ALP by 6 months that exceeded the least significant change of 25%. The bone ALP decrease from baseline exceeded 25% in 72 (97%) by the end of the study.Conclusion: The bone ALP assay is a sensitive and reliable tool that may be used to monitor the reduction in bone turnover after alendronate therapy in individual postmenopausal osteoporotic women.
Stereospecific Analysis and Enantiomeric Disposition of 3,4-Methylenedioxymethamphetamine (Ecstasy) in Humans
Authors: Fallon John K , Kicman Andrew T , Henry John A , Milligan Peter J , Cowan David A , Hutt Andrew J 
Published: January 2020

Abstract
Background: Little is known concerning the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) in humans. In addition, the potential of utilizing the stereochemical composition of an analyte in biological media for forensic purposes requires investigation.
Methods: The enantiomers of MDMA and its demethylated metabolite, 3,4-methylenedioxyamphetamine (MDA), present in plasma and urine extracts were derivatized with (−)-(R)-α-methoxy-α-trifluoromethylphenylacetyl chloride and analyzed by gas chromatography–mass spectrometry and gas chromatography, respectively. The enantioselective disposition of MDMA and MDA was determined following oral administration of racemic MDMA (40 mg) to eight male volunteers.
Results: The plasma concentrations of (R)-MDMA exceeded those of the S-enantiomer [ratio R:S of the area under the curve (AUC), 2.4 ± 0.3], and the plasma half-life of (R)-MDMA (5.8 ± 2.2 h) was significantly longer than that of the S-enantiomer (3.6 ± 0.9 h). The majority of the recovered material in urine was excreted within 24 h after dosing, with the recovery of (R)-MDMA (21.4% ± 11.6%) being significantly greater than that of (S)-MDMA (9.3% ± 4.9%), and with (S)- and (R)-MDA accounting for 1.4% ± 0.5% and 1.0% ± 0.3% of the dose, respectively. Mathematical modeling of plasma enantiomeric composition vs sampling time demonstrated the applicability of using stereochemical data for the prediction of time elapsed after drug administration.
Conclusions: Analytical methods for determining the enantiomeric composition of MDMA and MDA in plasma and urine were developed. The disposition of MDMA in humans is stereoselective, with the more active S-enantiomer having a reduced AUC and shorter half-life than (R)-MDMA. The determination of stereochemical composition may be applicable for forensic purposes.