scholarly journals Human plasma dynamically quenches the fluorescein at the initial point of Oxygen Radical Absorption Capacity (ORAC) Assay

2019 ◽  
Author(s):  
Harshi Prasadini Gunawardena ◽  
Renuka Silva ◽  
Pathmasiri Ranasinghe
Keyword(s):  
Human Plasma ◽  
Fluorescence Probe ◽  
Initial Point ◽  
Oxygen Radical ◽  
Absorption Capacity ◽  
Peroxyl Radicals ◽  
Dynamic Quenching ◽  
Initial Stage ◽  
First Time ◽  
Dose Dependent

Abstract Objective: Oxygen Radical Absorbance Capacity (ORAC) assay measures the quenching of fluorescent probe by peroxyl radicals. Antioxidants present in biological systems block the quenching of fluorescence probe. We experienced dynamic quenching of fluorescein in ORAC assay by human plasma while plasma ORAC assay was optimized. Therefore, for the first time, we report the quenching of fluorescein by plasma at the initial point of ORAC assay. Results: Aqueous whole and non-protein fractions of plasma were used in the analysis. Since both fractions showed a similar pattern of quenching at the initial stage, quenched percentage of fluorescein was calculated and added to each sample in subsequent analysis. Addition of extra 20% fluorescein allowed plasma samples to quench the required amount of fluorescein and follow normal decay curves afterwards. Further, change of fluorescein quenching (ΔF/F 0 ) disclosed a dose dependent linear relationship with plasma (R 2 =0.8). It can be speculated that dynamic quenching exhibited by human plasma biomolecule/s at the initial stage would be of non-protein aqueous phase molecule/s. We suggest initiating further studies to detect, identify and quantify the fluorescein quenching biomolecules present in human plasma for further improvements in plasma ORAC assay.

scholarly journals Human plasma dynamically quenches the fluorescein at the initial point of oxygen radical absorption capacity (ORAC) assay

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Harshi Gunawardena ◽  
Renuka Silva ◽  
Pathmasiri Ranasinghe
Keyword(s):  
Human Plasma ◽  
Fluorescence Probe ◽  
Initial Point ◽  
Oxygen Radical ◽  
Absorption Capacity ◽  
Peroxyl Radicals ◽  
Dynamic Quenching ◽  
Initial Stage ◽  
First Time ◽  
Dose Dependent

Abstract Objective Oxygen radical absorbance capacity (ORAC) assay measures the quenching of fluorescent probe by peroxyl radicals. Antioxidants present in biological systems block the quenching of fluorescence probe. We experienced the dynamic quenching of fluorescein, the fluorescence probe used in ORAC assay by the human plasma while plasma ORAC assay was optimized. Therefore, for the first time, we report the quenching of fluorescein by human plasma at the initial point of ORAC assay. Results Aqueous whole and non-protein fractions of plasma were used in the analysis. Since the both fractions showed a similar pattern of quenching at the initial stage, quenched percentage of fluorescein was calculated and added to each sample in subsequent analysis. Addition of extra 20% fluorescein allowed plasma samples to quench the required amount of fluorescein and follow the normal decay curves afterwards. Further, change of fluorescein quenching (ΔF/F0) disclosed a dose dependent linear relationship with plasma (R2 = 0.8). It can be speculated that dynamic quenching exhibited by human plasma biomolecule/s at the initial stage would be of non-protein aqueous phase molecule/s. We suggest initiating further studies to detect, identify and quantify the fluorescein quenching biomolecules present in human plasma for further improvements in plasma ORAC assay.

scholarly journals Human plasma dynamically quenches the fluorescein at the initial point of Oxygen Radical Absorption Capacity (ORAC) Assay

2019 ◽  
Author(s):  
Harshi Prasadini Gunawardena ◽  
Renuka Silva ◽  
Pathmasiri Ranasinghe
Keyword(s):  
Human Plasma ◽  
Fluorescence Probe ◽  
Initial Point ◽  
Oxygen Radical ◽  
Absorption Capacity ◽  
Peroxyl Radicals ◽  
Dynamic Quenching ◽  
Initial Stage ◽  
First Time ◽  
Dose Dependent

Abstract Objective: Oxygen Radical Absorbance Capacity (ORAC) assay measures the quenching of fluorescent probe by peroxyl radicals. Antioxidants present in biological systems block the quenching of fluorescence probe. We experienced the dynamic quenching of fluorescein, the fluorescence probe used in ORAC assay by human plasma while the plasma ORAC assay was optimized. Therefore, for the first time, we report the quenching of fluorescein by human plasma at the initial point of ORAC assay. Results: Aqueous whole and non-protein fractions of plasma were used in the analysis. Since both fractions showed a similar pattern of quenching at the initial stage, quenched percentage of fluorescein was calculated and added to each sample in subsequent analysis. Addition of extra 20% fluorescein allowed plasma samples to quench the required amount of fluorescein and follow the normal decay curves afterwards. Further, change of fluorescein quenching (ΔF/F0) disclosed a dose dependent linear relationship with plasma (R2=0.8). Conclusion: It can be speculated that dynamic quenching exhibited by human plasma biomolecule/s at the initial stage would be of non-protein aqueous phase molecule/s. We suggest initiating further studies to detect, identify and quantify the fluorescein quenching biomolecules present in human plasma for further improvements in plasma ORAC assay.

Determination of Total Antioxidant Capacity by Oxygen Radical Absorbance Capacity (ORAC) Using Fluorescein as the Fluorescence Probe: First Action 2012.23

2013 ◽  
Vol 96 (6) ◽  
pp. 1372-1376 ◽  
Author(s):  
Boxin Ou ◽  
Tony Chang ◽  
Dejian Huang ◽  
Ronald L Prior
Keyword(s):  
Antioxidant Capacity ◽  
Total Antioxidant Capacity ◽  
Water Solubility ◽  
Fluorescence Probe ◽  
Oxygen Radical ◽  
Oxygen Radical Absorbance Capacity ◽  
Peroxyl Radicals ◽  
Phenolic Antioxidants ◽  
Total Antioxidant

Abstract An improved method for the measurement of oxygen radical absorbance capacity (ORAC) was developed and validated using fluorescein (3′,6′-dihydroxyspiro[isobenzofuran-1[3H], 9′[9H]-xanthen]-3-one) as a new fluorescence probe (ORACFL). Randomly methylated β-cyclodextrin (RMCD) was introduced as the water-solubility enhancer for lipophilic antioxidants. 7% RMCD (w/v) in 50% acetone–H2O mixture sufficiently solubilized vitamin E compounds and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (pH 7.4). Results indicated that fluorescein shows excellent photostability under the plate reader conditions. This ORACFL was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrated that the ORACFL assay is reliable and robust. The mean of intraday and interday CVs were <15%; for hydrophilic ORAC, LOD and LOQ are 5 and 6.25 μM, respectively; for lipophilic ORAC, LOD and LOQ are 6.25 and 12.5 μM, respectively. It is concluded that unlike other popular methods, the ORACFL assay provides a direct measure of total antioxidant capacity against the peroxyl radicals.

scholarly journals Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 621
Author(s):  
Aurélien Millet ◽  
Nihel Khoudour ◽  
Jérôme Guitton ◽  
Dorothée Lebert ◽  
François Goldwasser ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Plasma ◽  
High Resolution Mass Spectrometry ◽  
Internal Standard ◽  
Therapeutic Drug ◽  
Limit Of Quantification ◽  
Immunoglobulin G4 ◽  
Positive Mode ◽  
Surrogate Peptide ◽  
First Time

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1–100 μg/mL, a limit of quantification at 1 μg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

scholarly journals Constituents of Chamaecrista diphylla (L.) Greene Leaves with Potent Antioxidant Capacity: A Feature-Based Molecular Network Dereplication Approach

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 681
Author(s):  
Paulo Gomes ◽  
Luis Quirós-Guerrero ◽  
Abraão Muribeca ◽  
José Reis ◽  
Sônia Pamplona ◽  
...  
Keyword(s):  
Antioxidant Capacity ◽  
Ethanolic Extract ◽  
Oxygen Radical ◽  
Molecular Network ◽  
Antioxidant Compounds ◽  
Herbaceous Plant ◽  
Network Approach ◽  
Feature Based ◽  
Promising Source ◽  
First Time

Chamaecrista diphylla (L.) Greene (Fabaceae/Caesalpiniaceae) is a herbaceous plant that is widely distributed throughout the Americas. Plants from this genus have been used in traditional medicine as a laxative, to heal wounds, and to treat ulcers, snake and scorpion bites. In the present study, we investigated the chemical composition of Chamaecrista diphylla leaves through a mass spectrometry molecular network approach. The oxygen radical absorbance capacity (ORAC) for the ethanolic extract, enriched fractions and isolated compounds was assessed. Overall, thirty-five compounds were annotated for the first time in C. diphylla. Thirty-two of them were reported for the first time in the genus. The isolated compounds 9, 12, 24 and 33 showed an excellent antioxidant capacity, superior to the extract and enriched fractions. Bond dissociation energy calculations were performed to explain and sustain the antioxidant capacity found. According to our results, the leaves of C. diphylla represent a promising source of potent antioxidant compounds.

scholarly journals Bearing Severity Fault Evaluation Using Contour Maps—Case Study

2021 ◽  
Vol 11 (14) ◽  
pp. 6452
Author(s):  
César Ricardo Soto-Ocampo ◽  
Juan David Cano-Moreno ◽  
José Manuel Mera ◽  
Joaquín Maroto
Keyword(s):  
Health Indicators ◽  
Bearing Faults ◽  
Contour Maps ◽  
Initial Stage ◽  
Critical Components ◽  
Bearing Damage ◽  
First Time ◽  
Current Operating

Increasing industrial competitiveness has led to an increased global interest in condition monitoring. In this sector, rotating machinery plays an important role, where the bearing is one of the most critical components. Many vibration-based signal treatments are already being used to identify features associated with bearing faults. The information embedded in such features are employed in the construction of health indicators, which allow for evaluation of the current operating status of the machine. In this work, the use of contour maps to represent the diagnosis map of a bearing, used as a health map, is presented for the first time. The results show that the proposed method is promising, allowing for the satisfactory detection and evaluation of the severity of bearing damage. In this initial stage of the research, our results suggest that this method can improve the classification of bearing faults and, therefore, optimise maintenance processes.

scholarly journals Human plasma fibronectin as a substrate for human urokinase

1989 ◽  
Vol 262 (2) ◽  
pp. 529-534 ◽  
Author(s):  
L I Gold ◽  
R Schwimmer ◽  
J P Quigley
Keyword(s):  
Extracellular Matrix ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Early Event ◽  
Plasma Fibronectin ◽  
Proteolytic Digestion ◽  
Enzyme Substrate ◽  
Tumour Cell Invasion ◽  
Substrate Ratio ◽  
Dose Dependent

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.

scholarly journals Intermittent Hypoxia Affects the Spontaneous DifferentiationIn Vitroof Human Neutrophils into Long-Lived Giant Phagocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Larissa Dyugovskaya ◽  
Slava Berger ◽  
Andrey Polyakov ◽  
Peretz Lavie ◽  
Lena Lavie
Keyword(s):  
Nadph Oxidase ◽  
Intermittent Hypoxia ◽  
Oxidase Inhibitor ◽  
Human Neutrophils ◽  
Hypoxic Conditions ◽  
Oxygen Conditions ◽  
Extended Lifespan ◽  
First Time ◽  
Dose Dependent

Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ) which spontaneously developin vitrowithout additional growth factors or cytokines. Gϕare CD66b+/CD63+/MPO+/LC3B+and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕformation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished Gϕformation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phoxexpression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phoxresembled Gϕformed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕformation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b+/LC3B+Gϕand increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕdevelopment.

scholarly journals High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 500-507 ◽  
Author(s):  
RN Puri ◽  
F Zhou ◽  
CJ Hu ◽  
RF Colman ◽  
RW Colman
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Plasma Concentration ◽  
Human Plasma ◽  
Shape Change ◽  
Dependent Manner ◽  
Normal Human Plasma ◽  
High Molecular Weight Kininogen ◽  
Normal Human ◽  
Dose Dependent

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)- induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin- induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma- thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma- thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

scholarly journals p38δ genetic ablation protects female mice from anthracycline cardiotoxicity

2020 ◽  
Author(s):  
Sharon A George ◽  
Alexi Kiss ◽  
Sofian N Obaid ◽  
Aileen Venegas ◽  
Trisha Talapatra ◽  
...  
Keyword(s):  
Cardiac Injury ◽  
Mapk Signaling ◽  
Anthracycline Cardiotoxicity ◽  
Genetic Ablation ◽  
Female Mice ◽  
Echocardiographic Parameters ◽  
The Individual ◽  
And Function ◽  
First Time ◽  
Dose Dependent

ABSTRACTBACKGROUNDThe efficacy of an anthracycline antibiotic doxorubicin (DOX) as a chemotherapeutic agent is limited by dose-dependent cardiotoxicity. DOX is associated with activation of intracellular stress signaling pathways including p38 MAPKs. While previous studies have implicated p38 MAPK signaling in DOX-induced cardiac injury, the roles of the individual p38 isoforms, specifically, of the alternative isoforms p38γ and p38δ, remain uncharacterized.OBJECTIVESTo determine the potential cardioprotective effects of p38γ and p38δ genetic deletion in mice subjected to acute DOX treatment.METHODSMale and female wild-type (WT), p38γ-/-, p38δ-/- and p38γ-/-δ-/- mice were injected with 30 mg/kg DOX and their survival was tracked for ten days. During this period cardiac function was assessed by echocardiography and electrocardiography and fibrosis by PicroSirius Red staining. Immunoblotting was performed to assess the expression of signaling proteins and markers linked to autophagy.RESULTSSignificantly improved survival was observed in p38δ-/- female mice post-DOX relative to WT females, but not in p38γ-/- or p38γ-/-δ-/- male or female mice. The improved survival in DOX-treated p38δ-/- females was associated with decreased fibrosis, increased cardiac output and LV diameter relative to DOX-treated WT females, and similar to saline-treated controls. Structural and echocardiographic parameters were either unchanged or worsened in all other groups. Increased autophagy, as evidenced by increased LC3-II level, and decreased mTOR activation was also observed in DOX-treated p38δ-/- females.CONCLUSIONSp38δ plays a crucial role in promoting DOX-induced cardiotoxicity in female mice by inhibiting autophagy. Therefore, p38δ targeting could be a potential cardioprotective strategy in anthracycline chemotherapy.NEW AND NOTEWORTHYThis study for the first time identifies the roles of the alternative p38γ and p38δ MAPK isoforms in promoting DOX-cardiotoxicity in a sex-specific manner. While p38γ systemic deletion did not affect DOX-cardiotoxicity, p38δ systemic deletion was cardioprotective in female but not in male mice. Cardiac structure and function were preserved in DOX-treated p38δ-/- females and autophagy was increased.

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