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1.
Int J Mol Sci ; 24(19)2023 Sep 28.
Article in English | MEDLINE | ID: mdl-37834149

ABSTRACT

Fluorescence lifetime measurements of blood or plasma offer valuable insights into the microenvironment and molecular interactions of fluorophores, particularly concerning albumin. Neutrophil- and hypoxia-induced oxidative stress in COVID-19 pneumonia patients leads to hyperinflammation, various oxidative modifications of blood proteins, and potential alterations in the fluorescence lifetime of tryptophan-containing proteins, especially albumin. The objective of this study was to investigate the efficacy of time-resolved fluorescence spectroscopy of blood and plasma as a prompt diagnostic tool for the early diagnosis and severity assessment of COVID-19-associated pneumonia. This study examined a cohort of sixty COVID-19 patients with respiratory symptoms. To investigate whether oxidative stress is the underlying cause of the change in fluorescence lifetime, human serum albumin was treated with chloramine T. The time-resolved spectrometer Life Spec II (Edinburgh Instruments Ltd., Livingston, UK), equipped with a sub-nanosecond pulsed 280 nm diode, was used to measure the fluorescence lifetime of blood and plasma. The findings revealed a significant reduction in the fluorescence lifetime of blood (diluted 200 times) and plasma (diluted 20 times) at 360 nm in COVID-19 pneumonia patients compared with their respective values recorded six months post-infection and those of healthy individuals. Significant negative correlations were observed between the mean fluorescence lifetime of blood and plasma at 360 nm and several severity biomarkers and advanced oxidation protein products, while a positive correlation was found with albumin and the albumin-globulin ratio. The time-resolved fluorescence spectroscopy method demonstrates the potential to be used as a preliminary screening technique for identifying patients who are at risk of developing severe complications. Furthermore, the small amount of blood required for the measurements has the potential to enable a rapid fingerstick blood test.


Subject(s)
COVID-19 , Humans , COVID-19/complications , COVID-19/diagnosis , Spectrometry, Fluorescence/methods , Blood Proteins , Albumins , Inflammation , COVID-19 Testing
2.
Int J Mol Sci ; 24(19)2023 Oct 06.
Article in English | MEDLINE | ID: mdl-37834401

ABSTRACT

Several studies have indicated that COVID-19 can lead to alterations in blood rheology, including an increase in red blood cell aggregation. The precise mechanisms behind this phenomenon are not yet fully comprehended. The latest findings suggest that erythrocyte aggregation significantly influences microcirculation, causes the formation of blood clots in blood vessels, and even damages the endothelial glycocalyx, leading to endothelial dysfunction. The focus of this research lies in investigating the cellular factors influencing these changes in aggregation and discussing potential causes and implications in the context of COVID-19 pathophysiology. For this purpose, the aggregation of erythrocytes in a group of 52 patients with COVID-19 pneumonia was examined in a 70 kDa Dextran solution, which eliminates the influence of plasma factors. Using image analysis, the velocities and sizes of the formed aggregates were investigated, determining their porosity. This study showed that the process of erythrocyte aggregation in COVID-19 patients, independent of plasma factors, leads to the formation of more compact, denser, three-dimensional aggregates. These aggregates may be less likely to disperse under circulatory shear stress, increasing the risk of thrombotic events. This study also suggests that cellular aggregation factors can be responsible for the thrombotic disorders observed long after infection, even when plasma factors have normalized. The results and subsequent broad discussion presented in this study can contribute to a better understanding of the potential complications associated with increased erythrocyte aggregation.


Subject(s)
COVID-19 , Erythrocyte Aggregation , Humans , Dextrans , Erythrocytes/physiology , Plasma
3.
Sci Rep ; 12(1): 19751, 2022 11 17.
Article in English | MEDLINE | ID: mdl-36396711

ABSTRACT

The aim of this study was to investigate the aggregation of red blood cells (RBCs) suspended in dextran solution at various levels of molecular mass. Dextran solutions at molecular mass 40, 70, 100 and 500 kDa at concentration from 2 to 5 g/dL were used to suspend the RBCs. The radius and velocity of sedimenting RBC aggregates were investigated using image analysis. The radius and sedimentation velocity of aggregates increased initially, then decreased after achieving maxima. The maximal velocity of RBC aggregates showed a bell-shaped dependence on dextran molecular mass and concentration, whereas maximal radius showed monotonic increase with both factors. Difference between aggregate and solution density was estimated using aggregate radius and sedimentation velocity and dextran solution viscosity, and was consistent across most molecular mass and concentration levels. This allowed to calculate the porosity of aggregates and to show that it monotonically decreased with the increase in the solution density, caused by the increase in the dextran concentration. The results provide insight into the RBC aggregation process in solutions of proteins of different size, reflecting various pathological conditions. The currently reported data can be potentially applied to specific pathophysiological conditions giving an interpretation that is not yet fully discussed in the literature.


Subject(s)
Dextrans , Erythrocyte Aggregation , Erythrocyte Aggregation/physiology , Molecular Weight , Erythrocytes , Erythrocyte Count
4.
Int J Mol Sci ; 23(17)2022 Sep 03.
Article in English | MEDLINE | ID: mdl-36077496

ABSTRACT

Oxidative stress induced by neutrophils and hypoxia in COVID-19 pneumonia leads to albumin modification. This may result in elevated levels of advanced oxidation protein products (AOPPs) and advanced lipoxidation end-products (ALEs) that trigger oxidative bursts of neutrophils and thus participate in cytokine storms, accelerating endothelial lung cell injury, leading to respiratory distress. In this study, sixty-six hospitalized COVID-19 patients with respiratory symptoms were studied. AOPPs-HSA was produced in vitro by treating human serum albumin (HSA) with chloramine T. The interaction of malondialdehyde with HSA was studied using time-resolved fluorescence spectroscopy. The findings revealed a significantly elevated level of AOPPs in COVID-19 pneumonia patients on admission to the hospital and one week later as long as they were in the acute phase of infection when compared with values recorded for the same patients 6- and 12-months post-infection. Significant negative correlations of albumin and positive correlations of AOPPs with, e.g., procalcitonin, D-dimers, lactate dehydrogenase, aspartate transaminase, and radiological scores of computed tomography (HRCT), were observed. The AOPPs/albumin ratio was found to be strongly correlated with D-dimers. We suggest that oxidized albumin could be involved in COVID-19 pathophysiology. Some possible clinical consequences of the modification of albumin are also discussed.


Subject(s)
Advanced Oxidation Protein Products , COVID-19 , Advanced Oxidation Protein Products/metabolism , Albumins/metabolism , Humans , Oxidation-Reduction , Oxidative Stress
5.
J Clin Med ; 11(17)2022 Aug 30.
Article in English | MEDLINE | ID: mdl-36079011

ABSTRACT

A method of rapidly pointing out the risk of developing persistent pulmonary fibrosis from a sample of blood is extraordinarily needed for diagnosis, prediction of death, and post-infection prognosis assessment. Collagen scar formation has been found to play an important role in the lung remodeling following SARS-CoV-2 infection. For this reason, the concentration of collagen degradation products in plasma may reflect the process of lung remodeling and determine the extent of fibrosis. According to our previously published results of an in vitro study, an increase in the concentration of type III collagen degradation products in plasma resulted in a decrease in the fluorescence lifetime of plasma at a wavelength of 450 nm. The aim of this study was to use time-resolved fluorescence spectroscopy to assess pulmonary fibrosis, and to find out if the lifetime of plasma fluorescence is shortened in patients with COVID-19. The presented study is thus far the only one to explore the fluorescence lifetime of plasma in patients with COVID-19 and pulmonary fibrosis. The time-resolved spectrometer Life Spec II with the sub-nanosecond pulsed 360 nm EPLED® diode was used in order to measure the fluorescence lifetime of plasma. The survival analysis showed that COVID-19 mortality was associated with a decreased mean fluorescence lifetime of plasma. The AUC of mean fluorescence lifetime in predicting death was 0.853 (95% CI 0.735−0.972, p < 0.001) with a cut-off value of 7 ns, and with 62% sensitivity and 100% specificity. We observed a significant decrease in the mean fluorescence lifetime in COVID-19 non-survivors (p < 0.001), in bacterial pneumonia patients without COVID-19 (p < 0.001), and in patients diagnosed with idiopathic pulmonary fibrosis (p < 0.001), relative to healthy subjects. Furthermore, these results suggest that the development of pulmonary fibrosis may be a real and serious problem in former COVID-19 patients in the future. A reduction in the mean fluorescence lifetime of plasma was observed in many patients 6 months after discharge. On the basis of these data, it can be concluded that a decrease in the mean fluorescence lifetime of plasma at 450 nm may be a risk factor for mortality, and probably also for pulmonary fibrosis in hospitalized COVID-19 patients.

6.
Sci Rep ; 12(1): 9012, 2022 05 30.
Article in English | MEDLINE | ID: mdl-35637245

ABSTRACT

The aim of this study was to examine the usefulness of time-resolved fluorescence spectroscopy in the evaluation of the oxidative processes in human plasma. To investigate the impact of oxidative stress on the fluorescence of plasma, five studied markers (thiobarbituric acid-reactive substances, ischemia modified albumin, carbonyl groups, hydrogen peroxide, advanced oxidation protein products) were chosen as oxidative damage approved markers. Our method presents several advantages over traditional methods as it is a direct, non-time-consuming, repeatable, and non-invasive technique that requires only simple pre-treatment of samples without additional reagents and the sample size needed for analysis is small. In principle, each modification of the protein in plasma can be expected to modify its fluorescence properties and hence its lifetime or intensity. The study involved 59 blood donors with no evidence of disease. The research was conducted at excitation wavelengths of 280 nm and 360 nm, and emission was measured at wavelengths of 350 nm and 440 nm, respectively. Our results, although preliminary, suggest that the application of fluorescence measurements can be considered as an effective marker of oxidative stress. Regression analyses showed that a notable growth in fluorescence intensity at 440 nm and a simultaneous decrease in fluorescence intensity and mean fluorescence lifetime at 350 nm are associated with higher levels of oxidative stress.


Subject(s)
Oxidative Stress , Serum Albumin , Biomarkers , Humans , Oxidation-Reduction , Spectrometry, Fluorescence/methods
7.
Molecules ; 27(2)2022 Jan 10.
Article in English | MEDLINE | ID: mdl-35056747

ABSTRACT

Ginkgo biloba is a popular medicinal plant widely used in numerous herbal products, including food supplements. Due to its popularity and growing economic value, G. biloba leaf extract has become the target of economically motivated adulterations. There are many reports about the poor quality of ginkgo products and their adulteration, mainly by adding flavonols, flavonol glycosides, or extracts from other plants. In this work, we developed an approach using two-trace two-dimensional correlation spectroscopy (2T2D COS) in UV-Vis range combined with multilinear principal component analysis (MPCA) to detect potential adulteration of twenty G. biloba food supplements. UV-Vis spectral data are obtained for 80% methanol and aqueous extracts in the range of 245-410 nm. Three series of two-dimensional correlation spectra were interpreted by visual inspection and using MPCA. The proposed relatively quick and straightforward approach successfully differentiated supplements adulterated with rutin or those lacking ginkgo leaf extract. Supporting information about adulteration was obtained from the difference between the DPPH radical scavenging capacity of both extracts and from chromatographic (HPLC-DAD) fingerprints of methanolic samples.


Subject(s)
Dietary Supplements/analysis , Food Contamination/analysis , Ginkgo biloba/chemistry , Spectrophotometry, Ultraviolet/methods , Cheminformatics/methods , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Kaempferols/analysis , Poland , Principal Component Analysis , Quercetin/analysis , Rutin/analysis
8.
Gen Physiol Biophys ; 37(6): 647-655, 2018 Sep.
Article in English | MEDLINE | ID: mdl-30338760

ABSTRACT

The aim of this study was to investigate the effect of oxidative stress on drug binding affinity, free and bound fraction. The fluorescence anisotropy method was used to examine the interactions of commercial human serum albumin (HSA) and human plasma proteins with ochratoxin A (OTA) or carboxylate form of camptothecin (CPT-C). In-vitro method revealed significant reduction in bound fraction of drugs to HSA oxidized by chloramine T. Oxidative stress was determined by measuring the plasma level of advanced oxidation protein products (AOPP). A significant positive correlation between the AOPP and the plasma free fraction of tested drugs in thirty healthy patients was also found. The oxidative stress monitoring by AOPP is very important for improvement of dosage of drugs highly bound to albumin and with narrow therapeutic index. As a result of severe oxidative stress, the drug pharmacokinetics and therapeutic effects are prone to change. This study highlights the issues of therapeutic drug monitoring and it can explain the behaviour of drugs in pathological situations.


Subject(s)
Oxidative Stress , Blood Proteins , Fluorescence Polarization , Humans , Oxidation-Reduction
9.
Acta Pol Pharm ; 73(1): 29-34, 2016.
Article in English | MEDLINE | ID: mdl-27008798

ABSTRACT

The study can be useful for understanding the interaction of camptothecin with human serum albu- min. There are two forms of camptothecin (the carboxylate form (CPT-C) and the lactone form (CPT-L)) but only the lactone one is pharmacologically active. It was reported earlier that in the presence of HSA, the active lactone form of camptothecin changes to inactive carboxylate form and it reduces the antitumor activities of camptothecin. However, those studies were performed at physiological pH (7.4) and with non-oxidized and non-glycosylated albumin. The aim of this study was to investigate the effect of oxidative stress, glycosylation, pH changes and competitor drugs on inactivation of lactone form of camptothecin in albumin solution using measurements of fluorescence anisotropy spectroscopy. It was tried to prove that in vivo camptothecin may be present in higher amount in lactone form than previously thought. Due to a reduction of pH value, a decreased rate of hydrolysis from CPT-L to CPT-C was observed. It was found in vitro a significant reduction in bound fraction of CPT-C to HSA oxidized by chloramine T or glycosylated by glucose. Moreover, as a result of block- ing binding of CPT-C to HSA by competitive compound (flurbiprofen), a decrease in the fluorescence anisotropy of the HSA-CPT complex was found. This study opens the way to review an application of CPT and its derivatives in therapy.


Subject(s)
Camptothecin/chemistry , Serum Albumin/chemistry , Fluorescence Polarization , Hydrogen-Ion Concentration , Spectrometry, Fluorescence
10.
Comb Chem High Throughput Screen ; 19(4): 319-24, 2016.
Article in English | MEDLINE | ID: mdl-26953237

ABSTRACT

Camptothecin (CPT) and its analogs as inhibitors of topoisomerase I are anticancer compounds. Their antitumor potency is seriously limited due to hydrolysis of lactone form of camptothecins in solutions at pH>5.5, which leads to the formation of inactive carboxylate form with open lactone ring. Furthermore, the clinical application of CPT is also restricted by strong affinity of its carboxylate form to human serum albumin which destabilizes the active lactone form. By UV irradiation of the CPT carboxylate authors of this paper received camptothecin compound which has biophysical properties similar to the lactone form. The specific objective of the project is to determine the properties using the methods of absorption and steady-state fluorescence spectra analysis, fluorescence lifetime measurements as well as steady-state fluorescence anisotropy. The results suggest that the UV exposed camptothecin carboxylate changes the chemical structure. The high-throughput assays based on the steady state fluorescence anisotropy measurements proved that the form obtained as a result of UV irradiation of CPT carboxylate exhibits weaker affinity to albumin than CPT carboxylate however stronger than CPT lactone. This property is very desirable from the point of view of clinical applications.


Subject(s)
Camptothecin/analogs & derivatives , Ultraviolet Rays , Camptothecin/chemistry , Camptothecin/metabolism , Camptothecin/radiation effects , Carboxylic Acids/metabolism , DNA Topoisomerases, Type I , Fluorescence Polarization , Humans , Lactones/metabolism , Serum Albumin/metabolism , Spectrometry, Fluorescence , Topoisomerase I Inhibitors
11.
J Biomed Opt ; 20(5): 051039, 2015 May.
Article in English | MEDLINE | ID: mdl-25764396

ABSTRACT

The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs' characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The in vitro model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial.


Subject(s)
Collagen/chemistry , Spectrometry, Fluorescence/methods , Animals , Collagen Type III/chemistry , Colorimetry , Female , Fluorescent Dyes/chemistry , Humans , Hydrolysis , Ninhydrin/chemistry , Placenta/metabolism , Pregnancy , Time Factors , Ventricular Remodeling
12.
Biosystems ; 94(3): 258-62, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18721856

ABSTRACT

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.


Subject(s)
Camptothecin/chemistry , Flurbiprofen/metabolism , Serum Albumin/metabolism , Warfarin/metabolism , Binding, Competitive , Fluorescence Polarization , Humans , Protein Binding
13.
Biosystems ; 94(3): 270-5, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18718502

ABSTRACT

Fluorescence spectroscopy methods are applied to the study of camptothecin analogue DB-67 and its ester DB-67-4ABTFA (trifluoroacetic acid salt of 20(S)-aminobutyrate substituted DB-67). Camptothecin and many of its analogues exhibit anticancer properties. They are fluorescent compounds, so using the method of fluorescence anisotropy measurements and fluorescence spectra recording many biophysical properties can be determined including affinity to proteins and membranes. One can also observe the process of conversion of the ester into DB-67. Active lactone form of camptothecin in fluids at pH 7.4 hydrolyses and converts into inactive carboxylate. Process of camptothecin deactivation is accelerated in plasma and after about 2h the total conversion to carboxylate form occurs. It is caused by fast and irreversible binding of carboxylate form to the human serum albumin (HSA). Camptothecin carboxylate bound to HSA does not lactonise. On the other hand, camptothecin lactone binding to membranes is reversible, but as long as lactone form bound to membranes does not hydrolyse. Knowledge of binding properties to proteins and membranes permits to select among many camptothecin analogues the ones exhibiting desirable behavior in physiological conditions: high affinity of lactone form to membranes and low affinity of carboxylate form to albumin. The studied DB-67 and DB-67-4ABTFA fulfill these requirements.


Subject(s)
Camptothecin/analogs & derivatives , Esters/chemistry , Organosilicon Compounds/chemistry , Protein Binding , Biophysics , Camptothecin/chemistry , Camptothecin/metabolism , Cell Membrane/metabolism , Esters/metabolism , Organosilicon Compounds/metabolism , Serum Albumin/metabolism , Spectrometry, Fluorescence
14.
Comb Chem High Throughput Screen ; 10(6): 459-65, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17896941

ABSTRACT

Camptothecins (CPTs) are fluorescent compounds exhibiting anticancer activity. They can exist in two forms, a lactone and a carboxylate. In neutral and base solution, lactone forms hydrolyse and convert into carboxylates. Only the lactone forms of CPTs are biologically active. Because of strong affinity of the carboxylate form of the parent drug camptothecin to human serum albumin (HSA), this protein promotes the deactivation of this compound. On the other hand, the lactone forms of camptothecins do not hydrolyse and are stabilized when bound to membranes. The following three hydroxycamptothecins, 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxy-camptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67) were studied. Factor analysis of a set of fluorescence excitation spectra recorded during lactone hydrolysis facilitated the high-throughput determination of the deactivation rates of camptothecin and each hydroxycamptothecin in phosphate buffered saline. The fluorescence spectra of hydroxycamptothecins diluted in HSA solution or suspended in DMPC liposomes were recorded, and the association constants of these drugs to membranes and plasma proteins were calculated. Among the analysed agents, DB-67 exhibited the most desirable properties including low affinity of the carboxylate form for albumin and high affinity of its lactone form for model membranes.


Subject(s)
Camptothecin/analogs & derivatives , Camptothecin/chemistry , Models, Biological , Serum Albumin/metabolism , Antineoplastic Agents, Phytogenic/chemistry , Camptothecin/metabolism , Humans , Irinotecan , Kinetics , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membranes, Artificial , Protein Binding , Serum Albumin/chemistry , Spectrometry, Fluorescence
15.
Comb Chem High Throughput Screen ; 10(6): 486-92, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17896945

ABSTRACT

Camptothecin (CPT) and its hydroxycamptothecin analogs are fluorescent compounds exhibiting strong anticancer properties. They exist in two forms: active lactone and inactive carboxylate. The deactivation proceeds via hydrolysis in neutral and base solutions. A serious limitation to the clinical application of CPT is the strong affinity of its carboxylate form to human serum albumin (HSA) which destabilizes its active lactone form. However, binding to membranes in blood improves the stability of the lactone form of CPT and its analogs. A high-throughput screening assay based on the steady-state fluorescence anisotropy method was used to determine the protein- and membrane-binding properties of 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67). The relative affinities of hydroxycamptothecins to HSA and model membranes in the form of DMPC liposomes were determined, and DB-67 exhibited the most desirable properties including the highest affinity to membranes in its lactone form and low affinity to HSA in its carboxylate form.


Subject(s)
Camptothecin/analogs & derivatives , Membranes/chemistry , Serum Albumin/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/metabolism , Camptothecin/chemistry , Camptothecin/metabolism , Fluorescence Polarization , Humans , Membranes/metabolism , Molecular Structure , Serum Albumin/metabolism
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