bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pitfalls in the Measurement of Circulating Vascular Endothelial Growth Factor
Authors: Jelkmann Wolfgang 
Published: January 2020

AbstractBackground: Vascular endothelial growth factor (VEGF) is a protein with antiapoptotic, mitogenic, and permeability-increasing activities specific for vascular endothelium. VEGF mRNA, which has five isoforms, is produced by nonmalignant cells in response to hypoxia and inflammation and by tumor cells in constitutively high concentrations. Because VEGF plays a crucial role in physiological and pathophysiological angiogenesis, measurements of circulating VEGF are of diagnostic and prognostic value, e.g., in cardiovascular failures, inflammatory diseases, and malignancies. However, there are major quantitative differences in the published results. This review attempts to identify reasons for these disparities.Approach: The literature was reviewed through a Medline search covering 1995 to 2000. A selection of exemplary references had to be made for this perspective overview.Content: Data are included from studies on healthy humans, gynecological patients, and persons suffering from inflammatory or malignant diseases. The results indicate that competitive immunoassays detect the total amount of circulating VEGF, which enables observations regarding the increase in VEGF in pregnancy and preeclampsia to be made. In these cases, capture immunoassays utilizing neutralizing antibodies are insufficient because of an accompanying increase in VEGF-binding soluble receptors (sFlt-1). Measurements of circulating free VEGF are useful for study of malignant diseases, which are associated with both genetically and hypoxia-induced overproduction of VEGF. The VEGF isoform specificity of the antibodies is also critical because both VEGF121 and VEGF165 are secreted. It is important to consider that platelets and leukocytes release VEGF during blood clotting.Conclusions: Future efforts should concentrate on the balance between free VEGF, total VEGF, and sFlt-1. Plasma, rather than serum, should be used for analysis.
Carcinoembryonic Antigen as a Marker for Colorectal Cancer: Is It Clinically Useful?
Authors: Duffy Michael J 
Published: January 2020

AbstractBackground: Carcinoembryonic antigen (CEA) is one of the most widely used tumor markers worldwide. Its main application is mostly in gastrointestinal cancers, especially in colorectal malignancy. Although in use for almost 30 years, the clinical value of CEA in colorectal cancer is still not clear.Methods: The literature relevant to the clinical value of CEA in colorectal cancer was reviewed. Particular attention was paid to studies involving metaanalyses and guidelines issued by Expert Panels.Results: Although of little use in detecting early colorectal cancer, high preoperative concentrations of CEA correlate with adverse prognosis. Serial CEA measurements can detect recurrent colorectal cancer with a sensitivity of ∼80%, a specificity of ∼70%, and can provide a lead time of ∼5 months. CEA is the most frequent indicator of recurrence in asymptomatic patients and currently is the most cost-effective test for the preclinical detection of resectable disease. CEA is most useful for the early detection of liver metastasis in patients with diagnosed colorectal cancer. Overall, however, little evidence is available that monitoring of all patients with diagnosed colorectal cancer leads to enhanced patient outcome or quality of life.Conclusions: Currently, the most useful application of CEA is in the detection of liver metastasis from colorectal cancers. Because of the relative success of surgery in resecting hepatic metastases, serial determinations of the marker are recommended for detecting cancer spread to the liver. In the future, preoperative concentrations of CEA may be included with the standard staging procedures for assessing prognosis.
DNA Melting Analysis for Detection of Single Nucleotide Polymorphisms
Authors: Lipsky Robert H , Mazzanti Chiara M , Rudolph Joseph G , Xu Ke , Vyas Gopal , Bozak David , Radel Marta Q , Goldman David 
Published: January 2020

Abstract
Background: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle.
Methods: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion).
Results: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15–167 bp in length and differing by only a single nucleotide substitution.
Conclusions: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.
Specific Reverse Transcription-PCR Quantification of Vascular Endothelial Growth Factor (VEGF) Splice Variants by LightCycler Technology
Authors: Wellmann Sven , Taube Tillmann , Paal Krisztina , Graf v. Einsiedel Hagen , Geilen Wilhelm , Seifert Georg , Eckert Cornelia , Henze Günter , Seeger Karlheinz 
Published: January 2020

AbstractBackground: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors, and hematologic malignancies. The expression of isoforms of VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formation.Methods: We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to β2-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.Results: Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (&lt;0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF121 and VEGF165 constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF189 constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF121 and VEGF165 accounted for 94% (SD = 7.6%) and VEGF189 only 6% (SD = 3.7%).Conclusions: Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.
Highly Sensitive, Automated Immunoassay for Immunoglobulin Free Light Chains in Serum and Urine
Authors: Bradwell Arthur R , Carr-Smith Hugh D , Mead Graham P , Tang Lian X , Showell Paul J , Drayson Mark T , Drew Roger 
Published: January 2020

AbstractBackground: Bence Jones proteins or monoclonal immunoglobulin κ and λ free light chains (FLCs) are important markers for identifying and monitoring many patients with B-cell tumors. Automated immunoassays that measure FLCs in urine and serum have considerable clinical potential.Methods: Sheep antibodies, specific for FLCs, were prepared by immunization with pure κ and λ molecules and then adsorbed extensively against whole immunoglobulins. The antibodies were conjugated onto latex particles and used to assay κ and λ FLCs on the Beckman IMMAGETM protein analyzer.Results: The unconjugated antibodies showed minimal cross-reactivity with intact immunoglobulins or other proteins. With latex-conjugated antibodies, κ and λ FLCs could be measured in normal sera and most normal urine samples. Patients with multiple myeloma had increased concentrations of the relevant serum FLC, whereas both FLCs were increased in the sera of patients with systemic lupus erythematosus.Conclusions: We developed sensitive, automated immunoassays for κ and λ FLC measurements in serum and urine that should facilitate the assessment of patients with light chain abnormalities.
Measurement of Immunoreactive Angiotensin-(1–7) Heptapeptide in Human Blood
Authors: Nussberger Juerg , Brunner Dorette B , Nyfeler Juerg A , Linder Lilly , Brunner Hans R 
Published: January 2020

AbstractBackground: The renal enzyme renin cleaves from the hepatic α2-globulin angiotensinogen angiotensin-(1–10) decapeptide [Ang-(1–10)], which is further metabolized to smaller peptides that help maintain cardiovascular homeostasis. The Ang-(1–7) heptapeptide has been reported to have several physiological effects, including natriuresis, diuresis, vasodilation, and release of vasopressin and prostaglandins.Methods: To investigate Ang-(1–7) in clinical settings, we developed a method to measure immunoreactive (ir-) Ang-(1–7) in 2 mL of human blood and to estimate plasma concentrations by correcting for the hematocrit. A sensitive and specific antiserum against Ang-(1–7) was raised in a rabbit. Human blood was collected in the presence of an inhibitor mixture including a renin inhibitor to prevent peptide generation in vitro. Ang-(1–7) was extracted into ethanol and purified on phenylsilylsilica. The peptide was quantified by radioimmunoassay. Increasing doses of Ang-(1–7) were infused into volunteers, and plasma concentrations of the peptide were measured.Results: The detection limit for plasma ir-Ang-(1–7) was 1 pmol/L. CVs for high and low blood concentrations were 4% and 20%, respectively, and between-assay CVs were 8% and 13%, respectively. Reference values for human plasma concentrations of ir-Ang-(1–7) were 1.0–9.5 pmol/L (median, 4.7 pmol/L) and increased linearly during infusion of increasing doses of Ang-(1–7).Conclusions: Reliable measurement of plasma ir-Ang-(1–7) is achieved with efficient inhibition of enzymes that generate or metabolize Ang-(1–7) after blood sampling, extraction in ethanol, and purification on phenylsilylsilica, and by use of a specific antiserum.
Fluorescence-based Detection of the CETPTaqIB Polymorphism: False Positives with the TaqMan-based Exonuclease Assay Attributable to a Previously Unknown Gene Variant
Authors: Teupser Daniel , Rupprecht Wolfgang , Lohse Peter , Thiery Joachim 
Published: January 2020

AbstractBackground: Previous studies have shown an association between the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene with plasma CETP and HDL concentrations and the progression of coronary artery disease (CAD). The aim of the present study was to determine the performance of two new fluorescence-based detection systems in the analysis of the TaqIB genotype.Methods: CAD patients (n = 150) with known TaqIB genotype, as determined by restriction fragment length polymorphism (RFLP) analysis, were selected, including three groups of 50 patients, carrying the B1/1, B1/2, and B2/2 genotypes, respectively. The genotypes were also analyzed by fluorescence-based allele-specific TaqMan PCR and melting curve analysis (LightCycler). In addition, DNA sequencing was applied.Results: The TaqIB genotypes obtained by fluorescence analysis corresponded to those determined by RFLP analysis with the exception of three heterozygous patients (B1/2), who were misclassified as homozygous B2 carriers with the TaqMan system. Melting curve analysis of these samples demonstrated an additional melting point at 59.1 °C, which was also found in four patients homozygous for the B1 allele. DNA sequencing revealed a previously unknown C270T nucleotide exchange in intron 1 of the CETP gene, only nine base pairs from the TaqIB site.Conclusions: Determination of the TaqIB polymorphism with the TaqMan system led to misclassifications because of a previously unknown C270T polymorphism of the CETP gene. The base substitution was detected with the LightCycler because of the occurrence of an additional melting point. Our data indicate the importance of thorough evaluation of new gene analysis systems before using them on a routine basis.
Quantification of Cellular Acid Sphingomyelinase and Galactocerebroside β-Galactosidase Activities by Electrospray Ionization Mass Spectrometry
Authors: Zhou Xinfeng , Tureček František , Scott C Ronald , Gelb Michael H 
Published: January 2020

AbstractBackground: Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside β-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample.Methods: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions.Results: The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39–70 nmol · mg−1 · h−1 compared with 3.7–5.1 nmol · mg−1 · h−1 in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0–6.8 nmol · mg−1 · h−1 in cell lysates from healthy patients compared with 0.1–0.2 nmol · mg−1 · h−1 in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 μg) for both ASM and GCG.Conclusions: Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.
Oxidative Stress and Homocysteine in Coronary Artery Disease
Authors: Cavalca Viviana , Cighetti Giuliana , Bamonti Fabrizia , Loaldi Alessandro , Bortone Luana , Novembrino Cristina , De Franceschi Michela , Belardinelli Romualdo , Guazzi Maurizio D 
Published: January 2020

Abstract
Background: Oxidative stress is present in cardiovascular diseases (CVDs), and hyperhomocysteinemia, an independent risk factor for these diseases, may play a role by inducing production of oxygen free radicals.
Methods: To evaluate the possible role of homocysteine (Hcy) in inducing oxidative stress in coronary artery disease (CAD), plasma Hcy was measured in 68 consecutive cardiovascular patients, and plasma malondialdehyde (MDA), both free and total (free + bound), was measured in 40 patients with CAD (18 with chronic stable angina and 22 with unstable angina). As controls, we tested 70 healthy volunteers. Hcy was measured by an immunoenzymatic method and MDA, an index of lipid peroxidation, by gas chromatography–mass spectrometry.
Results: Plasma Hcy concentrations were significantly higher in cardiovascular patients than in controls (10.2 vs 8.9 μmol/L; P &lt;0.0002), with no significant difference between values in the stable and unstable angina subgroups. Similarly, total MDA was significantly higher in the CAD group than in the controls (2.6 vs 1.3 μmol/L; P &lt;0.00001), again with no significant difference between stable and unstable angina patients. By contrast, free MDA, which was significantly higher in the CAD patients than the controls (0.4 vs 0.2 μmol/L; P &lt;0.00001), was also significantly higher in the unstable than in the stable angina group (0.5 vs 0.3 μmol/L; P &lt;0.03). However, no correlation was observed among Hcy and free and total MDA.
Conclusions: Our findings show that a moderate increase of Hcy is associated with CVD but that Hcy at the detected values cannot be considered completely responsible for oxidative damage. That lipid peroxidation is involved in CAD is shown by our observation of significantly increased plasma free and total MDA concentrations compared with controls. Moreover, free MDA values discriminated between unstable and chronic stable angina, and could thus represent a new diagnostic tool.
Heparin-induced Release of Protein-bound Solutes during Hemodialysis Is an in Vitro Artifact
Authors: De Smet Rita , Van Kaer Jacqueline , Liebich Hartmut , Lesaffer Gerrit , Verstraete Alain , Dhondt Annemieke , Duym Phillippe , Lameire Norbert , Vanholder Raymond 
Published: January 2020

AbstractBackground: Several studies have pointed to a release of drugs or protein-bound solutes from their binding sites during heparinization. The effect is attributed to the metabolism of triglycerides to free fatty acids (FFAs), which compete with drugs for protein binding sites. This study evaluated the impact of intradialytic heparin on the free concentration of the uremic toxin p-cresol and on FFAs.Methods: Blood samples from hemodialysis (HD) patients, before and during HD, were collected with selected anticoagulation strategies. We assessed the effects of standing time, temperature, pH, and the addition of a lipase inhibitor, tetrahydrolipstatin (THL) to blood samples on the free p-cresol concentration. p-Cresol was analyzed by HPLC with fluorescence detection. We measured FFAs by gas chromatography, and the free fractions of added valproic acid and phenytoin were evaluated by fluorescence polarization immunoassay.Results: In blood samples (n = 22) not submitted to a specific treatment, free p-cresol increased from 9.9 ± 5.1 to 31.9 ± 22.3 μmol/L after 30 min of heparin HD (P &lt;0.001) and correlated significantly with FFAs (r = 0.80; P = 0.002; n = 12). There was no increase in free p-cresol during heparin-free HD (n = 6) and trisodium citrate HD (n = 9). In addition, p-cresol in ultrafiltrates (n = 3) did not correspond to the free p-cresol in heparinized blood, suggesting that the increase in free p-cresol was artifactual. The release of p-cresol in the test tube was enhanced by standing time (n = 6), sample temperature (n = 6), and alkaline pH (n = 6). Inhibition of lipase activity with THL prevented the increase of FFAs (n = 6) and the release of free p-cresol during HD (n = 22). These results were corroborated by the study of the free fraction of valproic acid (n = 6) and phenytoin (n = 6).Conclusions: The free concentrations of protein-bound solutes in plasma of heparinized patients are influenced by external factors that alter the lipase activity in the test tube. The free fraction does not increase during HD when lipase activity is neutralized at the time of blood sampling, so that previously reported increases are probably artifacts.
Biochemical Markers of Bone Turnover and Response of Bone Mineral Density to Intervention in Early Postmenopausal Women: An Experience in a Clinical Laboratory
Authors: Chailurkit La-or , Ongphiphadhanakul Boonsong , Piaseu Noppawan , Saetung Sunee , Rajatanavin Rajata 
Published: January 2020

AbstractBackground: Markers of bone formation and resorption may be useful as early indicators of response to therapy. Our aim in this study was to investigate the use of bone markers for monitoring of intervention for bone loss in early postmenopausal women and to assess the relationships between these markers and changes in bone mineral density (BMD).Methods: Subjects were randomly assigned to the following groups: a control group; a group receiving calcium alone; groups receiving calcium plus low or conventional doses of conjugated equine estrogen; and groups receiving calcium plus low or conventional doses of calcitriol. At baseline and at 1 and 3 months after intervention, we measured serum intact osteocalcin, serum N-terminal midfragment osteocalcin, serum C-terminal telopeptide of type I collagen (CTx), urinary deoxypyridinoline cross-links, and urinary CTx. The BMD of the lumbar spine and the femoral neck was measured at baseline and after 1 and 2 years of intervention.Results: No marker changed significantly in the control group except urinary CTx, which increased at 3 months. Serum CTx decreased in all regimens at 1 or 3 months of intervention. In addition, the changes of all markers at 3 months were inversely associated with the change in the BMD of the lumbar spine at 1 or 2 years (r = −0.144 to −0.314), whereas only the changes of bone resorption markers at 3 months were inversely correlated with the changes in femoral BMD at 1 or 2 years (r = −0.143 to −0.366).Conclusions: Biochemical markers of bone turnover appear to be of use in assessing early response to therapy. Bone resorption markers, especially serum CTx, are better indicators than bone formation markers for estimating the response to intervention in early postmenopausal women. However, the early changes in bone markers were weakly related to the later changes in BMD.
Diagnostic Accuracy of the Anti-Citrulline Antibody Assay for Rheumatoid Arthritis
Authors: Bizzaro Nicola , Mazzanti Giovanni , Tonutti Elio , Villalta Danilo , Tozzoli Renato 
Published: January 2020

Abstract
Background: Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease, but specific and practicable tests for its diagnosis are lacking. We evaluated the diagnostic accuracy of a new commercial ELISA in detecting anti-cyclic citrullinated peptide (CCP) antibodies for the diagnosis of RA.
Methods: Anti-CCP antibodies were determined in 330 serum samples: 98 from RA patients and 232 from controls, including patients with connective tissue diseases, other rheumatic diseases, viral infections, Lyme disease, autoimmune thyroiditis, cancer, and monoclonal gammopathy, and sex- and age-matched healthy subjects. Intra- and interassay CVs were 5–13% and 9–17%, respectively. Rheumatoid factor (RF) was also assayed in every sample, and results were compared to anti-CCP for sensitivity and specificity.
Results: At a cutoff value of 50 units, sensitivity was 41% (confidence interval, 31–50%) and specificity was 97.8% (95–100%). Anti-CCP-positive RA patients had a mean antibody concentration of 1100 units (range, 57–3419 units), and anti-CCP-negative RA patients and controls had mean values of 7.6 and 6.8 units, respectively (range, 1–39 units). The area under the ROC curve was 0.71 (95% confidence interval, 0.63–0.78). RF had a higher sensitivity (62%) and a lower specificity (84%) than anti-CCP. When the two antibodies were used together, specificity was 99.6%.
Conclusion: Anti-CCP antibody testing may be useful if performed concomitantly with RF assay to diagnose patients with suspected early RA.