Quantitation of the Cancer Marker LASA Using Immobilized Enzymes in a Semiautomated System

Cancer patients are found to have elevated serum levels of lipid-associated sialic acid (LASA). LASA measurement provides a valuable test for the diagnosis of human cancer. The currently used measurement methods are tedious and nonselective. Here we report a method based on immobilized enzyme minicolumns in a flow system for the analysis of this clinically important analyte. Three enzymes comprising two minicolumns are introduced online and the analyte in a precisely injected volume of 20 μL is made to pass through these immobilized enzyme columns. The final product of the reaction is detected amperometrically. Neuraminidase and NANA-aldolase are coimmobilized on controlled pore glass, followed by a column of pyruvate oxidase. The method was applied to the quantitation of LASA in the sera of normal individuals and patients with cancer of the esophagus. We observed no loss of activity of the immobilized enzymes after analyzing about 300 samples. The sample throughput was 30 samples per hour.

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Interrelationship of Electrolyte and Vitamins Profile in Patients Experiencing Tumor Lysis Syndrome

10.53350/pjmhs211571597 ◽

2021 ◽

Vol 15 (7) ◽

pp. 1597-1599

Author(s):

Khadija Mastoor ◽

Bushra Suhail ◽

Asma Inam ◽

Nada Azam ◽

Maria Amjad ◽

...

Keyword(s):

Cancer Patients ◽

Tumor Lysis Syndrome ◽

Elevated Serum ◽

Serum Levels ◽

Prospective Clinical Study ◽

Metabolic Derangement ◽

Tumor Lysis ◽

Normal Individuals ◽

Life Threatening ◽

Older Cancer Patients

Background:Tumor lysis syndrome is a metabolic derangement which is seen in patients with malignancy and receiving drugs for cancer treatment. It can arise in children or older cancer patients and is considered life threatening. Anticancer drug therapy is most commonly used method to treat cancer. Aim: To investigate the role of electrolytes and vitamins (A, C and E) in cancer patients suffering from tumor lysis syndrome during anticancer therapy. Study design: Prospective clinical study Methods: The study enrolled fifty diagnosed patients of Tumor lysis syndrome.Informed consent was taken from patients.Twenty patients, clinically healthy, age and sex-matched were selected as a control in the present study. 5cc blood was withdrawn from enrolled cases. The obtained samples were centrifuged at the speed of 4000-5000rpm for 10-15 minutes to obtain serum. The levels of Electrolytes (Na+, K+), and Vitamins A, C, E were estimated. Results: Study showed elevated serum levels of sodium (Na+) (28.26) in comparison withcontrol normal persons (21.26) and this is significant statistically (0.02<0.05). Serum Potassium levels among Tumor lysis syndrome (TLS) cases was (13.26) as observed in normal controlled persons (14.26) and results were significant statistically (0.03<0.05). Vitamin A level in Tumor lysis syndrome(TLS) cases decreased outstandingly (102.20) in contrast to normal control study persons.(188.26) and this is significant statistically (0.026<0.05). The values for Vitamin E in Tumor lysis syndrome cases was (4.26) and in controlled normal individuals (7.26) and proved significant statistically (0.015<0.05). Conclusion: Present study showed inverse relationship between Vitamins and electrolytes in TLS. Increased level of electrolyte imbalances and decreased vitamin levels is the reason responsible for the development of tumor lysis syndrome. Keywords: TLS, Vit A, Vit C, Vit E, Na+,K+

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Serum MEPE-ASARM-peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets

Journal of Endocrinology ◽

10.1677/joe.1.05989 ◽

2004 ◽

Vol 183 (3) ◽

pp. R1-R9 ◽

Cited By ~ 81

Author(s):

Doron Bresler ◽

Jan Bruder ◽

Klaus Mohnike ◽

William D Fraser ◽

Peter S N Rowe

Keyword(s):

Elevated Serum ◽

Fold Increase ◽

Normal Subjects ◽

Serum Levels ◽

Hypophosphatemic Rickets ◽

Normal Male ◽

Renal Tubules ◽

Mineral Density ◽

Normal Individuals ◽

Asarm Peptide

MEPE (Matrix Extracellular PhosphoglycoprotEin) expression is markedly elevated in X-linked-hypophosphatemic-rickets (HYP) and tumor-induced osteomalacia (TIO). In normal individuals, circulating serum-levels of MEPE are tightly correlated with serum-phosphorus, parathyroid hormone (PTH) and bone mineral density (BMD). Also, MEPE derived, C-terminal ASARM-peptides are candidate minhibins and/or phosphatonins. Our aims were to determine: 1. whether MEPE-ASARM-peptide(s) are abnormally elevated in HYP/hyp serum, and, 2. whether the ASARM-peptide(s) accumulate in hyp mice kidney renal-tubules. Using a specific competitive ELISA we measured a five fold increase (P=0.007) of serum ASARM-peptide(s) in human HYP patients (normal subjects 3.25 μM n=9; s.e.m.=0.51 and HYP-patients 15.74 μM, n=9; s.e.m.=3.32). A 6.23 fold increase (P=0.008) was measured in hyp male mice compared with their normal male siblings (normal-siblings, 3.73 μM, s.e.m.=0.57, n=3; and hyp-mice 23.4 μM, n=3, s.e.m.=4.01). Renal immuno-histological screening also revealed a dramatic increase of ASARM-peptides in regions anatomically consistent with the proximal convoluted tubules. This study demonstrates for the first time that markedly elevated serum levels of protease-resistant ASARM-peptide(s) occur in HYP/hyp and they accumulate in murine hyp kidneys. These peptides are thus likely responsible for the phosphaturia and defective mineralization in HYP/hyp and TIO.

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Amperometric Determination of Total Cholesterol in Serum, with Use of Immobilized Cholesterol Ester Hydrolase and Cholesterol Oxidase

Clinical Chemistry ◽

10.1093/clinchem/23.4.671 ◽

1977 ◽

Vol 23 (4) ◽

pp. 671-676 ◽

Cited By ~ 33

Author(s):

Hua-shan Huang ◽

Shia S Kuan ◽

Guilbault George G

Keyword(s):

Serum Cholesterol ◽

Immobilized Enzyme ◽

Cholesterol Oxidase ◽

Immobilized Enzymes ◽

Total Serum ◽

Total Serum Cholesterol ◽

Amperometric Determination ◽

Standard Calomel Electrode ◽

Pass Through

Abstract We describe an electrochemical method for simple, rapid, and economical assay of total serum cholesterol with use of immobilized cholesterol esterase (EC 3.1.1.13) and cholesterol oxidase (EC 1.1.3.6). A rotating porous cell was specially designed to hold the immobilized enzymes firmly and to allow the reaction mixture to pass through the en¬zyme layer easily, thus catalyzing the enzymatic trans¬formation quickly. Hydrogen peroxide resulting from the catalytic reactions was measured amperometrically at +0.60 V vs. a standard calomel electrode. The calibration curve for total serum cholesterol was linear from 0 to 5.00 g/liter. The method is specific, precise, and inexpensive. Our results correlate well with those obtained by the method of Abell et al. [Stand. Methods Clin. Chem. 2, 26 (1958)] , the correlation coefficient being 0.992. Ascorbic acid or bilirubin in concentrations up to 100 mg/liter do not interfere. The immobilized enzymes are stable, and the same immobilized-enzyme stirrer can be used for at least 200 accurate, reproducible assays.

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Calorimetric Sensor for Ethanol Using Ni2+-nitrilotriacetic Acid (NTA) Resin Immobilized Alcohol Dehydrogenase (ADH)

Current Analytical Chemistry ◽

10.2174/1573411015666190617110233 ◽

2020 ◽

Vol 16 (6) ◽

pp. 795-799

Author(s):

YongJin Li

Keyword(s):

Cost Effective ◽

Nitrilotriacetic Acid ◽

Repeated Measurements ◽

Great Promise ◽

Calorimetric Sensor ◽

Controlled Pore Glass ◽

Pore Glass ◽

One Step ◽

Immobilization Matrix ◽

Enzyme Thermistor

Background: A simple, fast and economic analytical method for the determination of ethanol is important for clinical, biological, forensic and physico-legal purposes. Methods: Ni2+-NTA resin was used as an immobilization matrix for the simple one-step purification/ immobilization of his6-tagged ADH. Different alcohols with a concentration range of 0.5-50% V/V, namely methanol, ethanol and propanol were measured using prepared ADH enzyme thermistor. The ethanol content of Tsingtao beer was tested as a real sample containing alcohol. Reproducibility and stability of prepared ADH enzyme thermistor were also investigated by repeated measurements. Results: In comparison to the controlled pore glass (a common used support for the immobilization of enzyme) used in thermal biosensor, the use of Ni2+-NTA resin not only led to simple one-step purification/ immobilization by his6-tagged ADH binding to Ni2+-NTA resin, but also made the immobilizing supports reusable. The prepared biosensor can be used to determine ethanol and methanol by the calorimetric measurement. A linear range of 1 -32% (V/V) and 2-20% (V/V) was observed for ethanol and methanol, respectively. The detection limits were 0.3% (V/V) and 1% (V/V) for ethanol and methanol, respectively. The tested ethanol concentration of Tsingtao beer was 4.5% V/V, which is comparable with the labeled alcohol by volume (ABV) 4.80%. Conclusion: Ni2+-NTA resin, as an immobilization matrix in ET sensor, provides a simple one-step purification/immobilization for His6-tagged recombinase and a reusable immobilization matrix. The prepared biosensor exhibits good repeatability and stability. Such a new biosensor shows great promise for rapid, simple, and cost-effective analysis of ethanol and methanol, both in qualitative and in quantitative tests.

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Improved Conditions for Solid Phase Synthesis of Oligonucleotides on PS-PEG Copolymers

Zeitschrift für Naturforschung B ◽

10.1515/znb-1995-0720 ◽

1995 ◽

Vol 50 (7) ◽

pp. 1096-1100 ◽

Cited By ~ 14

Author(s):

Ernst Bayer ◽

Konrad Bleicher ◽

Martin Maier

Keyword(s):

Electrospray Mass Spectrometry ◽

Solid Phase Synthesis ◽

Solid Phase ◽

Chemical Properties ◽

Oligonucleotide Synthesis ◽

Phase Synthesis ◽

Controlled Pore Glass ◽

Product Identification ◽

Pore Glass ◽

And Chemical Properties

Polystyrene-polyethylene glycol (PS-PEG) tentacle polymers with loadings of up to 60/<μmol/g were used for standard oligonucleotide synthesis. As these resins are easy to handle and stable under reaction and cleavage conditions they may be used alternatively to controlled pore glass (CPG) as the most commonly used solid support for oligonucleotide synthesis. However, structural and chemical properties of the PS-PEG resins require modified conditions to guarantee syntheses with high coupling efficiencies. Oligonucleotides (ODN ) of various sequences and lengths have successfully been synthesized using HPLC and capillary electrophoresis (CE) for purity control. Additionally, electrospray mass spectrometry (ES-MS) was used for product identification.

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The Cytoskeletal Elements MAP2 and NF-L Show Substantial Alterations in Different Stroke Models While Elevated Serum Levels Highlight Especially MAP2 as a Sensitive Biomarker in Stroke Patients

Molecular Neurobiology ◽

10.1007/s12035-021-02372-3 ◽

2021 ◽

Author(s):

Bianca Mages ◽

Thomas Fuhs ◽

Susanne Aleithe ◽

Alexandra Blietz ◽

Constance Hobusch ◽

...

Keyword(s):

Animal Models ◽

Neuronal Damage ◽

Degradation Products ◽

Focal Cerebral Ischemia ◽

Elevated Serum ◽

Serum Levels ◽

Ultrastructural Level ◽

Stroke Patients ◽

Protein Levels ◽

Cytoskeletal Elements

AbstractIn the setting of ischemic stroke, the neurofilament subunit NF-L and the microtubule-associated protein MAP2 have proven to be exceptionally ischemia-sensitive elements of the neuronal cytoskeleton. Since alterations of the cytoskeleton have been linked to the transition from reversible to irreversible tissue damage, the present study investigates underlying time- and region-specific alterations of NF-L and MAP2 in different animal models of focal cerebral ischemia. Although NF-L is increasingly established as a clinical stroke biomarker, MAP2 serum measurements after stroke are still lacking. Therefore, the present study further compares serum levels of MAP2 with NF-L in stroke patients. In the applied animal models, MAP2-related immunofluorescence intensities were decreased in ischemic areas, whereas the abundance of NF-L degradation products accounted for an increase of NF-L-related immunofluorescence intensity. Accordingly, Western blot analyses of ischemic areas revealed decreased protein levels of both MAP2 and NF-L. The cytoskeletal alterations are further reflected at an ultrastructural level as indicated by a significant reduction of detectable neurofilaments in cortical axons of ischemia-affected areas. Moreover, atomic force microscopy measurements confirmed altered mechanical properties as indicated by a decreased elastic strength in ischemia-affected tissue. In addition to the results from the animal models, stroke patients exhibited significantly elevated serum levels of MAP2, which increased with infarct size, whereas serum levels of NF-L did not differ significantly. Thus, MAP2 appears to be a more sensitive stroke biomarker than NF-L, especially for early neuronal damage. This perspective is strengthened by the results from the animal models, showing MAP2-related alterations at earlier time points compared to NF-L. The profound ischemia-induced alterations further qualify both cytoskeletal elements as promising targets for neuroprotective therapies.

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Sustained high expression of multiple APOBEC3 cytidine deaminases in systemic lupus erythematosus

Scientific Reports ◽

10.1038/s41598-021-87024-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Danielle Perez-Bercoff ◽

Hélène Laude ◽

Morgane Lemaire ◽

Oliver Hunewald ◽

Valérie Thiers ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Cell Death ◽

Lupus Erythematosus ◽

Immunosuppressive Treatment ◽

Elevated Serum ◽

Serum Levels ◽

Chronic Inflammatory Disease ◽

Mrna Levels ◽

Systemic Lupus ◽

Inflammatory Conditions

AbstractAPOBEC3 (A3) enzymes are best known for their role as antiviral restriction factors and as mutagens in cancer. Although four of them, A3A, A3B, A3F and A3G, are induced by type-1-interferon (IFN-I), their role in inflammatory conditions is unknown. We thus investigated the expression of A3, and particularly A3A and A3B because of their ability to edit cellular DNA, in Systemic Lupus Erythematosus (SLE), a chronic inflammatory disease characterized by high IFN-α serum levels. In a cohort of 57 SLE patients, A3A and A3B, but also A3C and A3G, were upregulated ~ 10 to 15-fold (> 1000-fold for A3B) compared to healthy controls, particularly in patients with flares and elevated serum IFN-α levels. Hydroxychloroquine, corticosteroids and immunosuppressive treatment did not reverse A3 levels. The A3AΔ3B polymorphism, which potentiates A3A, was detected in 14.9% of patients and in 10% of controls, and was associated with higher A3A mRNA expression. A3A and A3B mRNA levels, but not A3C or A3G, were correlated positively with dsDNA breaks and negatively with lymphopenia. Exposure of SLE PBMCs to IFN-α in culture induced massive and sustained A3A levels by 4 h and led to massive cell death. Furthermore, the rs2853669 A > G polymorphism in the telomerase reverse transcriptase (TERT) promoter, which disrupts an Ets-TCF-binding site and influences certain cancers, was highly prevalent in SLE patients, possibly contributing to lymphopenia. Taken together, these findings suggest that high baseline A3A and A3B levels may contribute to cell frailty, lymphopenia and to the generation of neoantigens in SLE patients. Targeting A3 expression could be a strategy to reverse cell death and the generation of neoantigens.

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Dansylated Aminopropyl Controlled Pore Glass: A Model for Silica−Liquid Solvation+

Langmuir ◽

10.1021/la0490184 ◽

2004 ◽

Vol 20 (24) ◽

pp. 10507-10516 ◽

Cited By ~ 13

Author(s):

Phillip M. Page ◽

Chase A. Munson ◽

Frank V. Bright

Keyword(s):

Controlled Pore Glass ◽

Pore Glass

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Type 3 Deiodinase Role on Central Thyroid Hormone Action Affects the Leptin-Melanocortin System and Circadian Activity

Endocrinology ◽

10.1210/en.2016-1680 ◽

2016 ◽

Vol 158 (2) ◽

pp. 419-430 ◽

Cited By ~ 11

Author(s):

Zhaofei Wu ◽

M. Elena Martinez ◽

Donald L. St. Germain ◽

Arturo Hernandez

Keyword(s):

Energy Balance ◽

Elevated Serum ◽

Serum Levels ◽

Leptin Resistance ◽

Melanocortin System ◽

White Adipocyte ◽

Pituitary Thyroid Axis ◽

Thyroid Axis ◽

The Central Nervous System ◽

Type 3

Abstract The role of thyroid hormones (THs) in the central regulation of energy balance is increasingly appreciated. Mice lacking the type 3 deiodinase (DIO3), which inactivates TH, have decreased circulating TH levels relative to control mice as a result of defects in the hypothalamic-pituitary-thyroid axis. However, we have shown that the TH status of the adult Dio3−/− brain is opposite that of the serum, exhibiting enhanced levels of TH action. Because the brain, particularly the hypothalamus, harbors important circuitries that regulate metabolism, we aimed to examine the energy balance phenotype of Dio3−/− mice and determine whether it is associated with hypothalamic abnormalities. Here we show that Dio3−/− mice of both sexes exhibit decreased adiposity, reduced brown and white adipocyte size, and enhanced fat loss in response to triiodothyronine (T3) treatment. They also exhibit increased TH action in the hypothalamus, with abnormal expression and T3 sensitivity of genes integral to the leptin-melanocortin system, including Agrp, Npy, Pomc, and Mc4r. The normal to elevated serum levels of leptin, and elevated and repressed expression of Agrp and Pomc, respectively, suggest a profile of leptin resistance. Interestingly, Dio3−/− mice also display elevated locomotor activity and increased energy expenditure. This occurs in association with expanded nighttime activity periods, suggesting a disrupted circadian rhythm. We conclude that DIO3-mediated regulation of TH action in the central nervous system influences multiple critical determinants of energy balance. Those influences may partially compensate each other, with the result likely contributing to the decreased adiposity observed in Dio3−/− mice.

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