bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prolonged Hyperlipasemia Attributable to a Novel Type of Macrolipase
Authors: Taes Youri E , Louagie Henk , Yvergneaux Jean-Pierre , De Buyzere Marc L , De Puydt Hendrik , Delanghe Joris R , Lott John A 
Published: January 2020

Abstract
Background: We present the case of an 80-year-old woman who was admitted to hospital with an intermittent volvulus of the right colon. A total colectomy was performed. Initially, serum amylase and lipase increased concordantly, but after a few weeks amylase normalized (85 U/L), whereas lipase increased to 3764 U/L. This discrepancy and persistence of hyperlipasemia suggested a macromolecular form of lipase.
Methods: The nature of the macromolecular complex was studied using high-pressure liquid gel-permeation chromatography, affinity chromatography, (immuno)electrophoresis, and immunodiffusion.
Results: Gel-permeation chromatography revealed a macrolipase, with a molecular mass &gt;900 kDa, that contributed up to 56% of total serum lipase activity. Butanol extraction of the specimen did not alter the elution profile. The thermostabilities of pancreatic lipase and the macroform were similar, whereas activation energy (Ea) was lower in the macromolecular lipase (28 ± 4 kJ · mol−1 · K−1 vs 48 ± 7 kJ · mol−1 · K−1 (P = 0.02). Agarose electrophoresis showed a broad band of lipase activity at the application site. Protein A-Sepharose affinity gel chromatography excluded IgG-linked lipase. Agarose electrophoresis and immunofixation excluded linkage to other immunoglobulins. Radial immunodiffusion did not show lipase activity in the immunoglobulin precipitation bands. Radial immunodiffusion with α2-macroglobulin (α2-MG) antibodies showed a diffuse spot of lipase activity within the precipitation band, suggesting a macromolecular association between lipase and α2-MG. Affinity gel chromatography against α2-MG showed lipase activity in the α2-MG-bound fractions.
Conclusion: This is the first report of a macrolipase in which an association between α2-MG and lipase is described.
Improved Stable Isotope Dilution-Gas Chromatography-Mass Spectrometry Method for Serum or Plasma Free 3-Hydroxy-Fatty Acids and Its Utility for the Study of Disorders of Mitochondrial Fatty Acid β-Oxidation
Authors: Jones Patricia M , Quinn Rebecca , Fennessey Paul V , Tjoa Susan , Goodman Stephen I , Fiore Stephany , Burlina Alberto B , Rinaldo Piero , Boriack Richard L , Bennett Michael J 
Published: January 2020

Abstract
Background: Disorders of fatty acid oxidation (FAO) are difficult to diagnose, primarily because in many of the FAO disorders measurable biochemical intermediates accumulate in body fluids only during acute illness. Increased concentrations of 3-hydroxy-fatty acids (3-OH-FAs) in the blood are indicative of FAO disorders of the long- and short-chain 3-hydroxy-acyl-CoA dehydrogenases, LCHAD and SCHAD. We describe a serum/plasma assay for the measurement of 3-OH-FAs with carbon chain lengths from C6 to C16.
Methods: We used stable isotope dilution gas chromatography-mass spectrometry (GC-MS) with electron impact ionization and selected ion monitoring. Natural and isotope-labeled compounds were synthesized for the assay.
Results: The assay was linear from 0.2 to 50 μmol/L for all six 3-OH-FAs. CVs were 5–15% at concentrations near the upper limits seen in healthy subjects. In 43 subjects, the medians (and ranges) in μmol/L were as follows: 3-OH-C6, 0.8 (0.3–2.2); 3-OH-C8, 0.4 (0.2–1.0); 3-OH-C10, 0.3 (0.2–0.6); 3-OH-C12, 0.3 (0.2–0.6); 3-OH-C14, 0.2 (0.0–0.4); and 3-OH-C16, 0.2 (0.0–0.5). 3-OH-FAs were increased in infants receiving formula containing medium chain triglycerides. Two patients diagnosed with LCHAD deficiency showed marked increases in 3-OH-C14 and 3-OH-C16 concentrations. Two patients diagnosed with SCHAD deficiency showed increased shorter chain 3-OH-FAs but no increases in 3-OH-C14 to 3-OH-C16.
Conclusion: Measuring blood concentrations of the 3-OH-FAs with this assay may be a valuable tool for helping to rapidly identify deficiencies in LCHAD and SCHAD and may also provide useful information about the status of the FAO pathway.
Evaluation of a Bead-based Enzyme Immunoassay for the Rapid Detection of Osteocalcin in Human Serum
Authors: Crăciun Alexandra M , Vermeer Cees , Eisenwiener Hans-Georg , Drees Norbert , Knapen Marjo H J 
Published: January 2020

AbstractBackground: Circulating osteocalcin is a well-known marker for bone formation, but none of the commercial kits currently available can be used in automated systems. Here we present the first semiautomated assay for human serum osteocalcin.Methods: Polystyrene beads were coated with antibodies against the COOH terminus of osteocalcin and used in the COBAS® EIA System. Osteocalcin was detected with peroxidase-conjugated antibodies against the osteocalcin NH2 terminus.Results: The time required to analyze an unknown sample was 60 min, with a lower detection limit of 4.5 μg/L and a linear dose–response curve between 4.5 and 100 μg/L. The intraassay imprecision (CV) was 5–8% (n = 21); the interassay variation was 6–9% (n = 14). In samples from human volunteers and patients, data generated with the newly developed assay were comparable to those obtained with standard microtiter plate-based assays.Conclusions: The coated beads assay may be implemented on fully automated analyzers, which not only may further reduce imprecision but may also substantially increase the applicability of osteocalcin as a marker for bone metabolism in the routine clinical setting.
Measurement of Plasma and Intracellular S-Adenosylmethionine and S-Adenosylhomocysteine Utilizing Coulometric Electrochemical Detection: Alterations with Plasma Homocysteine and Pyridoxal 5′-Phosphate Concentrations
Authors: Melnyk Stepan , Pogribna Marta , Pogribny Igor P , Yi Ping , James S Jill 
Published: January 2020

AbstractBackground: The relative changes in plasma and intracellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) may be important predictors of cellular methylation potential and metabolic alterations associated with specific genetic polymorphisms and/or nutritional deficiencies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn processing or metabolite derivatization.Methods: We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 μL of plasma, 106 lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted isocratically.Results: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4–5.5% and the intraassay CV was 2.8–5.6%. The analytical recoveries were 96.8% and 97.3% for SAM and SAH, respectively. In a cohort of healthy adult women with mean total homocysteine concentrations of 7.3 μmol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concentrations (mean, 12.1 μmol/L), plasma SAH, but not SAM, was increased (P &lt;0.001), and plasma pyridoxal 5′-phosphate concentrations were reduced (P &lt;0.001). Plasma SAM/SAH ratios were inversely correlated with homocysteine concentrations (r = 0.73; P &lt;0.01), and the SAM/SAH ratio in plasma was directly correlated with the intracellular SAM/SAH ratio in lymphocytes (r = 0.70; P &lt;0.01).Conclusions: Increased homocysteine in serum is associated with an increase in SAH and a decrease in the SAM/SAH ratio that could negatively affect cellular methylation potential. Accurate and sensitive detection of these essential metabolites in plasma and in specific tissues should provide new insights into the regulation of one-carbon metabolism under different nutritional and pathologic conditions.
Correlation of Antemortem Serum Creatine Kinase, Creatine Kinase-MB, Troponin I, and Troponin T with Cardiac Pathology
Authors: Ooi Daylily S , Isotalo Phillip A , Veinot John P 
Published: January 2020

AbstractBackground: Spurious increases in serum troponins, especially troponin T, have been reported in patients with and without acute myocardial syndromes.Methods: We studied 78 autopsied patients without clinical myocardial infarction (MI) and correlated histologic cardiac findings with antemortem serum creatine kinase (CK), its MB isoenzyme (CK-MB), cardiac troponin I (cTnI), and cardiac troponin T (cTnT).Results: There was no significant myocardial pathology in 15 patients. Cardiac pathologies were in five groups: scarring from previous MI or patchy ventricular fibrosis (n = 9), recent MI (n = 27), healing MI (n = 7), degenerative myocyte changes consistent with congestive heart failure (CHF; n = 12), and other cardiac pathologies (n = 8). The median concentrations in the five groups were not significantly different for either CK or CK-MB. Compared with the no-pathology group, only the MI group was significantly different for cTnI, and the MI and other pathology groups were significantly different for cTnT. For patients with MI, 22%, 19%, 48%, and 65% had increased CK, CK-MB, cTnI, and cTnT, respectively; for CHF and other cardiac pathologies combined, the percentages were 28%, 17%, 22%, and 50%. For patients with increased cTnI, 72% and 28% had MI and other myocardial pathologies, respectively; patients with increased cTnT had 64% and 36%, respectively. Patients without myocardial pathology had no increases in CK-MB, cTnI, or cTnT.Conclusions: All patients with increased serum CK-MB, cTnI, and cTnT had significant cardiac histologic changes. The second-generation cTnT assay appears to be a more sensitive indicator of MI and other myocardial pathologies than the cTnI assay used in this study.
Determination of the Acyl Glucuronide Metabolite of Mycophenolic Acid in Human Plasma by HPLC and Emit
Authors: Shipkova Maria , Schütz Ekkehard , Armstrong Victor William , Niedmann Paul Dieter , Oellerich Michael , Wieland Eberhard 
Published: January 2020

AbstractBackground: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated.Methods: The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents.Results: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r &gt;0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99–103% for MPA, 95–103% for 7-O-MPAG, and 104–107% for AcMPAG. The within-day imprecision was &lt;5.0% for MPA (0.2–25 mg/L), &lt;4.4% for 7-O-MPAG (10–250 mg/L), and ≤14% for AcMPAG (0.1–5 mg/L). The between-day imprecision was &lt;6.2%, &lt;4.5%, and ≤14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1–10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 °C or −20 °C. Acidified samples (pH 2.5) were stable up to 30 days at −20 °C.Conclusions: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.
Detection and Measurement of Urinary 2-Hydroxyestradiol 17-Sulfate, a Potential Placental Antioxidant during Pregnancy
Authors: Takanashi Kaori , Honma Takashi , Kashiwagi Tomohiro , Honjo Hideo , Yoshizawa Itsuo 
Published: January 2020

Abstract
Background: Preeclampsia is associated with a quantitative imbalance between lipid peroxide and an antioxidant coproduced in the placenta. To investigate our hypothesis that 2-hydroxyestradiol 17-sulfate (2-OH-ES) is the placental antioxidant during pregnancy, we developed an assay for 2-OH-ES in urine and studied samples from women with and without preeclampsia.
Methods: The detection and measurement of 2-OH-ES in the urine of pregnant women were performed by RIA using highly specific antiserum to 2-OH-ES. To confirm the reliability of the RIA method, the same samples were analyzed by HPLC using an electrochemical detector.
Results: Urinary 2-OH-ES values obtained by RIA showed a close relationship to those obtained by HPLC (y = 1.1x − 0.01; r = 0.96). The urinary 2-OH-ES concentrations during the first, second, and third trimesters were 2.0 ± 0.6 (mean ± SE, n = 13), 5.3 ± 1.3 (n = 21), and 15.3 ± 2.0 μg/mg creatinine (n = 54), respectively, and &lt;0.15 μg/mg creatinine (n = 10) at 2–24 h after delivery. The concentrations in preeclamptic women during the third trimester were significantly lower, 3.9 ± 1.9 μg/mg creatinine (mean ± SE, n = 12).
Conclusions: RIA can be used to measure urinary 2-OH-ES during pregnancy. The increase in urinary 2-OH-ES during gestation, its decrease after delivery, and the lower values in preeclampsia are consistent with a role of 2-OH-ES as a placental antioxidant.
Leptin Binding and Binding Capacity in Serum
Authors: Landt Michael 
Published: January 2020

Abstract
Background: Leptin, a hormone produced primarily by adipose tissue, is known to be present in serum as both monomeric (free) and higher molecular mass (bound) forms, but little is known about the nature of the bound forms or physiological variation in binding capacity.
Methods: A new method to quantify the free and bound forms was developed, based on HPLC separation and RIA quantification in chromatography fractions. Reanalysis of specimens after addition of exogenous leptin allowed direct determination of leptin-binding capacity and the degree of saturation of leptin-binding capacity.
Results: HPLC chromatography fractionated serum leptin into both the free form and as a broad peak of 59–130 kDa. Several experiments were conducted to validate the new method. The concentrations of bound leptin in serum were 0.45–3.94 μg/L, and they increased as total leptin (reflecting adiposity) increased in 24 lean and obese volunteers. Leptin was readily dissociated from the bound fraction by competition from exogenous leptin. Rechromatography of the bound fraction led to dissociation of leptin, which was promoted by warming the sera before chromatography. Leptin-binding capacity was 1.8–5.3 μg/L; binding capacity was nearly constant over a range of total leptin concentrations of 2–10 μg/L, and slowly increased at higher total leptin concentrations. Saturation of binding capacity was low (15%) at very low total leptin concentrations (&lt;5 μg/L), but rose quickly to a plateau near 80% at higher total leptin concentrations.
Conclusions: The new method facilitates measurement of free and bound fractions of serum leptin, and is the first method measuring leptin-binding capacity. These experiments demonstrate that the concentration of bound leptin and leptin-binding capacity vary physiologically, with binding/binding capacity increasing with adiposity. Except in very lean individuals, binding capacity is nearly completely saturated.
Investigation by Isoelectric Focusing of the Initial Carbohydrate-deficient Transferrin (CDT) and non-CDT Transferrin Isoform Fractionation Step Involved in Determination of CDT by the ChronAlcoI.D. Assay
Authors: Hackler Rolf , Arndt Torsten , Helwig-Rolig Angelika , Kropf Juergen , Steinmetz Armin , Schaefer Juergen R 
Published: January 2020

Abstract
Background: The introduction of a new set of reagents for the determination of carbohydrate-deficient transferrin (CDT) as a marker of chronic alcohol abuse requires an independent evaluation of the analytic specificity of the test. This information is needed for correct interpretation and classification of test results.
Methods: Isoelectric focusing on the PhastSystemTM followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D.TM assay.
Results: The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV ≤10%). Except for quantitatively unimportant traces of trisialo-Fe2-transferrin (&lt;5% of total CDT), only asialo-, mono-, and disialo-Fe2-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe2-transferrin (during column rinsing) and disialo-Fe2-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe2-transferrin did not change from &gt;1 (serum) to &lt;1 (eluates) as described for the CDTect assays. The transferrin patterns in the ChronAlcoI.D. eluates were representative of those in serum. Transferrin D variants with isoelectric points close to that of trisialo-Fe2-transferrin C1 did not cause overdetermination of CDT by the ChronAlcoI.D. test.
Conclusions: The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.
Seasonal and Biological Variation of Blood Concentrations of Total Cholesterol, Dehydroepiandrosterone Sulfate, Hemoglobin A1c, IgA, Prolactin, and Free Testosterone in Healthy Women
Authors: Garde Anne Helene , Hansen Åse Marie , Skovgaard Lene Theil , Christensen Jytte Molin 
Published: January 2020

AbstractBackground: Concentrations of physiological response variables fluctuate over time. The present study describes within-day and seasonal fluctuations for total cholesterol, dehydroepiandrosterone sulfate (DHEA-S), hemoglobin A1c (HbA1c), IgA, prolactin, and free testosterone in blood, and estimates within- (CVi) and between-subject (CVg) CVs for healthy women. In addition, the index of individuality, prediction intervals, and power calculations were derived.Methods: A total of 21 healthy female subjects participated in the study. Using a random effects analysis of variance, we estimated CVg and total within-subject variation (CVti), i.e., the combined within-subject and analytical variation, from logarithmically transformed data. Analytical variation was subtracted from CVti to give CVi. CVi was estimated from samples taken monthly during 1 year (CViy), weekly during 1 month (CVim), and six times within 1 day (CVid).Results: A cyclic seasonal variation was demonstrated for total cholesterol, DHEA-S, HbA1c, prolactin, and free testosterone. Within-day variation was shown for prolactin and free testosterone. The overall mean values for the group and the variability (CViy and CVg) were: 5.1 mmol/L, 5.5%, and 5.0% for total cholesterol; 6.6 μmol/L, 7.1%, and 21% for DHEA-S; 4.3%, 2.6%, and 3.3% for HbA1c/hemoglobintotal; 2.1 g/L, 5.9%, and 13% for IgA; 136 mIU/L, 23%, and 27% for prolactin; and 5.4 pmol/L, 21%, and 29% for free testosterone.Conclusions: Collecting samples at specific hours of the day or times of the year may reduce high biological variation. Alternatively, the number of individuals may be increased and a paired study design chosen to obtain adequate statistical power.
Analysis of Concentration and 13C Enrichment of d-Galactose in Human Plasma
Authors: Schadewaldt Peter , Hammen Hans-Werner , Loganathan Kamalanathan , Bodner-Leidecker Annette , Wendel Udo 
Published: January 2020

Abstract
Background: A stable-isotope dilution method for the sensitive determination of d-galactose in human plasma was established.
Methods: d-[13C]Galactose was added to plasma, and the concentration was measured after d-glucose was removed from the plasma by treatment with d-glucose oxidase and the sample was purified by ion-exchange chromatography. For gas chromatographic–mass spectrometric analysis, aldononitrile pentaacetate derivatives were prepared. Monitoring of the [MH-60]+ ion intensities at m/z 328, 329, and 334 in the positive chemical ionization mode allowed the assessment of 1-12C-, 1-13C-, and U-13C6-labeled d-galactose, respectively. The d-galactose concentration was quantified on the basis of the 13C-labeled internal standard.
Results: The method was linear (range examined, 0.1–5 μmol/L) and of good repeatability in the low and high concentration ranges (within- and between-run CVs &lt;15%). The limit of quantification for plasma d-galactose was &lt;0.02 μmol/L. Measurements in plasma of postabsorptive subjects yielded d-galactose concentrations (mean ± SD) of 0.12 ± 0.03 (n = 16), 0.11 ± 0.04 (n = 15), 1.44 ± 0.54 (n = 10), and 0.17 ± 0.07 (n = 5) μmol/L in healthy adults, diabetic patients, patients with classical galactosemia, and obligate heterozygous parents thereof, respectively. These data were considerably lower (3- to 18-fold) than the values of a conventional enzymatic assay. The procedure was also applied successfully in a stable-isotope turnover study to evaluate endogenous d-galactose formation.
Conclusions: The present findings establish that detection of d-galactose from endogenous sources is feasible in human plasma and show that erroneously high results may be obtained by enzymatic methods.
DzyNA-PCR: Use of DNAzymes to Detect and Quantify Nucleic Acid Sequences in a Real-Time Fluorescent Format
Authors: Todd Alison V , Fuery Caroline J , Impey Helen L , Applegate Tanya L , Haughton Margaret A 
Published: January 2020

Abstract
Background: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel.
Methods: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts.
Results: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum.
Conclusion: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.