bluuebunny/crossref_metadata_2025_split huggingface dataset:
Preliminary Treatment of Urinary Proteins Improves Electrophoretic Analysis and Immunodetection
Authors: Verdier J M , Dussol B , Dupuy P , Berland Y , Dagorn J C 
Published: January 2020

Abstract
Analysis of urinary protein composition is an important tool in studies on renal physiology and physiopathology. Urine is, however, a complex mixture containing, besides protein, a variety of compounds such as salts, peptides, oligosaccharides, and glycosaminoglycans. Some of these compounds interfere with the electrophoretic migration of protein in sodium dodecyl sulfate-polyacrylamide gels and prevent correct analysis of the protein pattern. We describe a simple method for extracting urinary proteins that considerably improves their electrophoretic migration and subsequent immunodetection. This treatment involves ammonium sulfate fractionations (for precipitating proteins), EDTA (for inhibiting protein aggregation), and HCl hydrolysis (for removing glycosylaminoglycans). Recovery during extraction was found to be almost quantitative for total protein and three representative proteins: albumin, alpha 1-glycoprotein acid, and beta 2-microglobulin.
Intralaboratory Performance Requirements Necessary to Pass Proficiency Testing: CAP-1990 vs CLIA-1967 (March 14, 1990) Formats Compared
Authors: Laessig R H , Ehrmeyer S S , Leinweber J E 
Published: January 2020

Abstract
The pre-1990 College of American Pathologists' (CAP) Proficiency Testing (PT) program used a two samples per analyte/four challenges per year format with performance or pass-fail grading criteria determined by the program. On Jan. 1, 1991, the Clinical Laboratory Improvement Act of 1967 (CLIA-67) final rules (March 14, 1990) mandated a revised PT format of five samples per analyte/four challenges per year, with the regulations specifying minimum performance criteria. Extending our previous analysis, we compare the maximum permissible intralaboratory imprecision at low bias compatible with passing external PT in the former CAP and current CLIA-67 formats. If a laboratory is able to reduce its internal coefficient of variation (CV) to less than 44% of the PT criterion for each analyte, its overall chance of adverse action for any of the 27 routine chemistry analytes specified in CLIA-67 will be less than 1% in a two-year (eight PT challenges or events) period. Consideration of actual interlaboratory CVs from CAP surveys suggest that a reduction of this magnitude may be difficult for the analytes total cholesterol and blood urea nitrogen, where intralaboratory imprecision comparable with the group standard deviation (SD) from 1990 CAP surveys would yield individual adverse action (PT failure) rates of 5% and 1%, respectively. Five other analytes have CLIA-67 performance limits dangerously close to CAP interlaboratory CVs.
Failure of Common Glycation Assays to Detect Glycation by Fructose
Authors: Ahmed N , Furth A J 
Published: January 2020

Abstract
Serum albumin was modified by in vitro glycation with either fructose or glucose, to see whether the common clinical assays for glycation were able to detect both fructose- and glucose-induced changes in protein structure in diabetes. Although fluorescence measurements showed that fructose causes far more protein damage than glucose, neither serum fructosamine (SFA) nor phenylboronate affinity (PBA) glycation assays reflected these changes. The SFA method implied that fructose causes only about 5% of the glycation induced by glucose; with PBA the proportion was 25%. The thiobarbituric acid- and periodate-based assays also greatly underestimated the true extent of fructation. We discuss these discrepancies with respect to the underlying chemistry, emphasizing the difference between aldehydic and ketonic Amadori products (exemplified by fructose and glucose derivatives, respectively). The implications for detecting fructose-induced secondary diabetic complications are also discussed.
Effects of Serum Bilirubin on Determination of Uric Acid by the Uricase-Peroxidase Coupled Reaction
Authors: Aoki Y , Ihara H , Nakamura H , Aoki T , Yoshida M 
Published: January 2020

Abstract
We examined the possible effect of different bilirubin species, including unconjugated (Bu), sugar-conjugated (Bc), and authentic delta bilirubin (Bd) isolated from serum, on the direct enzymatic measurement of uric acid by using the uricase (EC 1.7.3.3) and peroxidase (EC 1.11.1.7) coupled reaction. Bc, isolated from human bile, produced the most interference with uric acid determination. Although the presence of human serum albumin reduced interference by Bu and Bc, albumin-bound Bc complex still produced greater interference than Bu. Interference with the peroxidase reaction by Bc covalently bound to serum albumin (Bd) was less than that by Bu. We examined this phenomenon by using serum-isolated Bd obtained by ammonium sulfate precipitation and ultrafiltration and by using commercially available ditaurobilirubin (DTB) and chemically synthesized Bd (via Woodward's reagent, WBd) as surrogates for bile-isolated Bc and natural Bd, respectively. DTB produced the same amount of interference as natural Bc, but the interference produced by DTB in the presence of serum albumin was not the same as that produced by natural Bc with albumin. Our synthetic Bd appears to be a reliable surrogate for natural Bd in testing for bilirubin interference.
Multiple Occurrence of Macro Creatine Kinase in One Family
Authors: Bayer P M , Kraus S 
Published: January 2020

Abstract
We measured creatine kinase (CK, EC 2.7.3.2), CK-MB isoenzyme activity and mass concentration, and distribution of CK isoenzymes (by electrophoresis) in serum from five members of one family. The mother and two young children showed CK-IgG complexes in their sera. The concentration of the CK-IgG complexes in the children decreased over time, suggesting that the complex involved maternal IgG and had been transferred across the placenta from the mother to her children.
Robotic Automation of Cyclosporine Analysis in Whole Blood
Authors: Holman J W , Felder R A 
Published: January 2020

Abstract
Cyclosporin A (CsA) is currently the most selective immunosuppressant used clinically to prevent organ rejection after transplantation. We used a reprogrammable robot, the Benchmate (Zymark Inc., Hopkinton, MA), to automate much of the sample preparation involved in solid-phase extraction of CsA. Lysed whole-blood specimens containing previously added internal standard were placed on the Benchmate in the specimen-holding area, and a C18 Bond-Elut column was placed in the top of the sample tube. Specimen extraction from this point was handled automatically by the Benchmate, after which we manually injected the sample into the HPLC system for quantification. Analytical recovery of CsA in two concentrations of calibrator was similar for the manual and Benchmate methods. Analytical imprecision for the Benchmate was less than for manual extraction: within-run imprecision (CV) was less than 8% at either concentration. Between-run imprecision, determined by having the Benchmate extract CsA from two specimens each day for 20 days, was less than 9%. Patients' specimens were extracted manually (x) or by using the Benchmate robot (y); the results, compared by linear regression, agreed well: (y = 0.99 (SD 0.02)x + 2.6 (SD 2.8) micrograms/L (Sy[x = 7.59 micrograms/L, n = 27). We conclude that the Benchmate usefully reduces the manual steps necessary to extract specimens before HPLC analysis for CsA.
Characterization of Hemoglobin Lepore Variants by Advanced Mass-Spectrometric Procedures
Authors: De Caterina M , Esposito P , Grimaldi E , Di Maro G , Scopacasa F , Ferranti P , Parlapiano A , Malorni A , Pucci P , Marino G 
Published: January 2020

Abstract
We describe an analytical protocol for characterizing the molecular structure of hemoglobin (Hb) Lepore variants by using two different mass-spectrometric approaches. The first method consists of direct examination of the chromatographically separated hybrid globins by electro-spray mass spectrometry; the variant Lepore globin is identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by fast atom bombardment mass-spectrometric analysis of the peptide mixture. The application of this procedure to the identification of Hb Lepore Boston and Hb Lepore Baltimore is described.
Electrophoretic Fractionation of Pancreatic Lipase
Authors: Panteghini M 
Published: January 2020

Abstract
Human pancreatic isolipases were separated by electrophoresis on cellulose acetate membrane. Band visualization by fluorescence detection of NADH produced by beta-oxidation cyclic reaction of fatty acid liberated from 1,2-dilinoleoylglycerol by lipase was achieved by making a two-plate "sandwich" of an electrophoresis plate and a substrate-bearing plate. Two true lipase forms, named L1 and L2 to reflect their electrophoretic migration, were resolved with this method and quantified by densitometry. The procedure was not only simple and rapid but also precise (between-run CV less than 13.5%) and sensitive (detected lipase fractions of less than 2 U/L). L1 (mean 57% of total lipase, SD 19.3%) and L2 (mean 43%, SD 19.3%) lipase forms were found in all normal persons studied. In a preliminary clinical study of 27 emergency room patients, evaluation of the lipase electrophoretic pattern in serum did not separate patients with acute pancreatitis from those with other acute abdominal disorders.
Direct Double Monoclonal Immunoradiometric Assay of Urinary Human Growth Hormone
Authors: Porquet D , Rigal O , Brion D E , Valade F , Leger J , Czernichow P 
Published: January 2020

Abstract
Several reports indicate that urinary growth hormone (GH) excretion might reflect central release of the hormone, and that measurement of urinary GH shows promise in the investigation of physiological and pathological GH secretion. We have developed and evaluated a direct immunoradiometric assay (IRMA) in which two monoclonal antibodies are used to measure GH in the urine of children. The detection limit is approximately 0.018 pmol/L for a sample volume of 2 mL. Within- and between-run variations (CVs) were 5.6% and 14.2%, respectively. Analytical recovery and dilution experiments showed the specificity of the method for GH. In normal-stature prepubertal children ages 3-12 years, 24-h urinary GH excretion was 0.189 (SD 0.100) pmol and correlated well with the amount of GH in the first morning miction, which showed wide day-to-day variations. Like others, we found a strong correlation between GH concentrations in serum and urine during stimulation tests with GH-releasing hormone (somatoliberin) and (or) during physiological nocturnal secretion, confirming that urinary GH measurement may be of help in investigating patients (particularly young children) with diseases in which GH secretion is impaired.
Sensitive and Selective Liquid-Chromatographic Assay of Fluoxetine and Norfluoxetine in Plasma with Fluorescence Detection After Precolumn Derivatization
Authors: Suckow R F , Zhang M F , Cooper T B 
Published: January 2020

Abstract
We determined fluoxetine (Prozac) and its major metabolite norfluoxetine in plasma by liquid chromatography with fluorescence detection. After liquid-liquid extraction from 1 mL of plasma, the extract was derivatized at room temperature with dansyl chloride, and the highly fluorescent derivatives were chromatographed with a reversed-phase C18 column and a mobile phase of phosphate buffer and acetonitrile. Dansylated fluoxetine, norfluoxetine, and the internal standard were eluted in less than 14 min with no interference from endogenous material. The calibration curve was linear over the concentration range 25-800 micrograms/L with inter- and intra-assay imprecision (CV) of less than 10%. Validity of the assay was checked by comparing results for 110 patients' samples with those by a liquid-chromatographic method with ultraviolet detection (r = 0.993 for fluoxetine, 0.957 for norfluoxetine). The identity of the dansylated derivatives was verified by positive chemical ionization mass spectroscopy. The lower limit of detection was approximately 3 micrograms/L. Because no major antidepressant, neuroleptic, or respective drug metabolites interfere with the quantification of fluoxetine and norfluoxetine, this is a useful procedure for pharmacokinetic studies and in clinical settings.
Presence of Angiotensin Peptides in Human Urine
Authors: Hermann K , Rittweger R , Phillips M I , Ring J 
Published: January 2020

Abstract
Immunoreactive angiotensin I and angiotensin II were found in human urine that was purified on octadecasilylsilica cartridges. The daily excretion of angiotensin I and II in healthy volunteers was 189.00 (SE 38.36) and 17.54 (SE 3.07) pmol/24 h or 148.09 (SE 32.22) and 12.82 (SE 2.34) pmol/L, respectively (n = 12). No circadian rhythm was observed in the excretion patterns of angiotensin I and II. In vitro degradation of angiotensin I or II could not be detected in acidified urine samples. A marked increase in the excretion of angiotensin I and II could be demonstrated in patients with anaphylactoid reactions to drugs and food additives after oral challenge. Immunoreactive angiotensin I and II could be characterized by HPLC as Ile5-angiotensin I, Ile5-angiotensin II, and angiotensin II metabolites.
Detection of Testosterone Administration by Increased Ratio Between Serum Concentrations of Testosterone and 17 Alpha-Hydroxyprogesterone
Authors: Carlström K , Palonek E , Garle M , Oftebro H , Stanghelle J , Björkhem I 
Published: January 2020

Abstract
An increased ratio between urinary testosterone (T) and epitestosterone (epiT) has been accepted by the International Olympic Committee as a marker for T doping. However, in a few subjects, we and others have observed constantly above-normal urinary T/epiT ratios that are unlikely to be related to exogenous T administration. To find a better test for T doping, we studied several serum and urinary androgens and androgen precursors, estrogens, and luteinizing hormone (LH) in seven healthy volunteers for 35 days after an intramuscular injection of 250 mg of testosterone enanthate. Among urinary analyses, only the T/epiT ratio was a suitable marker of T doping; of the serum assays, 17 alpha-hydroxyprogesterone (17OHP), T/17OHP ratio, LH, and T/LH ratio were fair to good markers of T doping. The serum T/17OHP ratio was the best marker of those tested, with all seven subjects having above-normal values for this in the first 3 days of the observation period. No other marker showed abnormal values in all subjects at any time. Moreover, the T/17OHP ratio was affected by neither diurnal variation nor physical stress. The value of this marker for T doping was further supported by the finding of normal T/17OHP ratios in a subject with increased urinary T/epiT ratios caused by an abnormally low testicular epiT production, probably related to genetic factors.
Simple Method for Quantifying Alpha-Tocopherol in Low-Density+Very-Low-Density Lipoproteins and in High-Density Lipoproteins
Authors: Carcelain G , David F , Lepage S , Bonnefont-Rousselot D , Delattre J , Legrand A , Peynet J , Troupel S 
Published: January 2020

Abstract
We assessed the distribution of alpha-tocopherol in serum lipoprotein samples after separating the lipoprotein fractions by either sequential ultracentrifugation or selective precipitation with sodium phosphotungstate-magnesium chloride reagent. alpha-Tocopherol concentrations were determined by reversed-phase high-performance liquid chromatography. After ultracentrifugation, we found that in men, low- and very-low-density serum lipoproteins (LDL-VLDL) contained 53.6% of alpha-tocopherol vs 46.4% in high-density lipoproteins (HDL). In women, serum LDL-VLDL contained 45.6% alpha-tocopherol after ultracentrifugation vs 54.4% in HDL. After selective precipitation, the proportions of alpha-tocopherol in men were 56.1% in LDL-VLDL vs 43.9% in HDL, and in women, 45.4% in LDL-VLDL vs 54.6% in HDL. After selective precipitation, alpha-tocopherol recovery from whole lipoprotein fractions was 97% to 100% vs 80% after ultracentrifugation, thus allowing more accurate alpha-tocopherol quantification than after separation by ultracentrifugation.
Detection of Antithyroglobulin Autoantibodies with Defined Epitopic Specificity by a Microparticle-Enhanced Nephelometric Immunoassay
Authors: Harchali A A , Montagne P , Cuillière M L , Bouanani M , Pau B , Duheille J 
Published: January 2020

Abstract
To hydrophilic, polyfunctional spherical microparticles of predetermined diameter, produced by copolymerization of acrylic monomers, we covalently bound human thyroglobulin. The thyroglobulin-microsphere conjugate was agglutinated, in the presence of antimouse immunoglobulins antiserum, by four monoclonal antibodies, each recognizing a different antigenic domain on the thyroglobulin molecule. These agglutinations were quantified by measuring with a specially designed nephelometer the light scattered by clusters of the conjugates. Agglutination with the monoclonal antibody recognizing antigenic domain II of the thyroglobulin molecule was specifically inhibited by some human sera that contained antithyroglobulin autoantibodies. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for these autoantibodies with defined epitopic specificity. Using this assay, we detected and quantified antithyroglobulin autoantibodies in serum samples from all eight patients examined with Hashimoto disease and from most (75%) patients with untreated Graves disease.
Quantification of beta 2-microglobulin and albumin in plasma and peritoneal dialysis fluid by a noncompetitive immunoenzymometric assay
Authors: Kandoussi A , Cachera C , Pagniez D , Saile R , Tacquet A 
Published: January 2020

Abstract
An enzyme-linked immunosorbent assay (ELISA) was developed for measuring beta 2-microglobulin (beta 2m) and albumin in continuous ambulatory peritoneal dialysis (CAPD) fluid. Plasma concentrations of beta 2m were twofold greater in hemodialysis patients (41.3 +/- 13.3 mg/L) than in CAPD patients (23.6 +/- 5.5 mg/L) matched for duration of treatment. Measurement of beta 2m in CAPD fluid showed a substantial loss of this protein, approximately 31% of total body beta 2m, compared with a 5% loss of a protein of middle molecular mass (albumin). Because of the molecular sieving effects of the peritoneal membrane, peritoneal clearance of beta 2m was sixfold greater than that of albumin. Whether beta 2m losses prevent or delay the incidence of dialysis-induced amyloidosis in these patients remains to be established.
Competitive assay to improve the specificity of detection of single-point mutations in alpha 1-antitrypsin deficiency
Authors: Gunneberg A , Scobie G , Hayes K , Kalsheker N 
Published: January 2020

Abstract
Allele-specific oligonucleotides are used widely for the detection of single point mutations in genes. A modification of this assay based on competition has been developed for detection of the Z mutation of alpha 1-antitrypsin (alpha 1-AT). The normal alpha 1-AT allele is referred to as M, and the Z mutation arises from a single base substitution. Amplified DNA products corresponding to homozygous M, heterozygous MZ, and homozygous Z obtained by the polymerase chain reaction were incubated with a twofold molar excess of unlabeled oligonucleotide prior to hybridization with a radiolabeled oligonucleotide. Thus, initial incubation with unlabeled M-specific oligonucleotide was followed by hybridization with radiolabeled Z-specific oligonucleotide, and vice versa. This assay increased the specificity of single-point mutation detection three- to four-fold. Furthermore, specific hybridization was obtained at a lower temperature as a consequence of improving the signal-to-noise ratio.
Simultaneous determination of vitamins A and E and carotenoids in plasma by reversed-phase HPLC in elderly and younger subjects
Authors: Zaman Z , Fielden P , Frost P G 
Published: January 2020

Abstract
A reversed-phase high-performance liquid-chromatographic method for the simultaneous determination of retinol, alpha-tocopherol, alpha-carotene, beta-carotene, cryptoxanthin, lutein/zeaxanthin, and lycopene is described. This method was applied to plasma measurements in healthy young and elderly subjects. The plasma, deproteinized with ethanol, is extracted twice with n-hexane. After evaporation, the residue is dissolved in 50 microL of tetrahydrofuran and made up to 200 microL with ethanol. Samples (50 microL) are injected onto a 250 x 4.6 mm column of 5-microns-particle Spherisorb ODS1 (Phase Separations) that had been equilibrated with solvent mixture A:B (90:10 by vol) [A = 100 mmol/L ammonium acetate in methanol: acetonitrile (80:20 by vol) and B = 100 mmol/L ammonium acetate in water] at 2 mL/min. The analytes are eluted by running a 12-min linear gradient to 100% A; solvent A is then maintained for 10 min. Intrabatch CVs were 2.3%, 3.3%, 2.8%, 3.6%, 3.6%, and 3.0% for retinol, alpha-tocopherol, lutein/zeaxanthin, cryptoxanthin, lycopene, and beta-carotene, respectively. The corresponding interbatch CVs were 4.9%, 5.8%, 12.3%, 6.5%, 8.0%, and 3.4%.
Evaluation of EMIT Cyclosporine Assay for use with whole blood
Authors: Beresini M H , Davalian D , Alexander S , Toton-Quinn R , Barnett B , Cerelli M J , Hu M W , Berger D E , Blohm W P , Jaklitsch A 
Published: January 2020

Abstract
We evaluated the EMIT Cyclosporine Assay, designed for specific quantification of cyclosporine (CsA) in whole blood. The assay was run on the Roche Cobas Mira or Cobas Mira S analyzer. Analytical recovery from both normal donor whole blood and transplant patients' samples was within 17% of the standards. Measurement of diluted out-of-range samples also yielded excellent recovery (101-105%). Within-run, between-run, and total CVs were &lt; or = 8.1%, 10.9%, and 12.1%, respectively. The detection limit was &lt; 32 micrograms/L. The assay was linear from 0 to 500 micrograms/L. No significant cross-reactivity was observed for CsA metabolites AM1, AM19, and AM4N. Slight cross-reactivity occurred with metabolite AM9; 670 micrograms/L AM9 increased the measured CsA concentration by 49 micrograms/L. High concentrations of bilirubin, uric acid, triglycerides, and cholesterol, as well as 54 commonly coadministered drugs did not interfere with CsA quantification. Similarly, neither extreme values of hematocrit nor choice of anticoagulant affected CsA recovery. Sample extract stability was &gt; 4 h, and assay calibration was stable for at least 2 weeks. Patients' samples analyzed by the EMIT assay showed strong correlation with both HPLC and 125I-RIA (r &gt; 0.97). We conclude that the EMIT Cyclosporine Assay provides a convenient, accurate, and precise method for specific quantification of CsA in whole blood.
HPLC assay of melatonin in plasma with fluorescence detection
Authors: Peniston-Bird J F , Di W L , Street C A , Kadva A , Stalteri M A , Silman R E 
Published: January 2020

Abstract
We report an HPLC assay for melatonin that incorporates automated injection, methanol/water mobile phase, and fluorescence detection. Plasma samples were extracted by solid and liquid phases. Recovery was &gt; 70% for 1-10 mL of plasma extracted, approximately 40 pg-250 ng of melatonin. Samples were dried and reconstituted in 100 mL/L methanol. Injections were 25 microL or 150 microL, depending on sample concentration, and the melatonin peak was eluted in 380 mL/L methanol. The detection limit of the assay was 6 pg on the column, allowing a practical sensitivity in plasma of 11 pmol/L for 8-mL samples and 34 pmol/L for 2-mL samples. More than 100 plasma samples from volunteers and patients were assayed and the results compared with an established RIA. The mean daytime concentration of melatonin was 20.7 pmol/L (SEM = 1.2) and 18.5 pmol/L (SEM = 1.6) for HPLC and RIA, respectively, and the mean nighttime concentration was 82.4 pmol/L (SEM = 6.5) and 82.2 (SEM = 7.3), respectively.