Biphasic dose-response and effects of near-infrared photobiomodulation on erythrocytes susceptibility to oxidative stress in vitro.

The effect of simultaneous application of tert-butyl hydroperoxide (tBHP) and polychromatic near-infrared (NIR) radiation on bovine blood was examined to determine whether NIR light decreases the susceptibility of red blood cells (RBCs) to oxidative stress. The study assessed various exposure methods, wavelength ranges, and optical filtering types. Continuous NIR exposure revealed a biphasic response in cell-free hemoglobin changes, with antioxidative effects observed at low fluences and detrimental effects at higher fluences. Optimal exposure duration was identified between 60 s and 15 min. Protective effects were also tested across wavelengths in the range of 750-1100 nm, with all of them reducing hemolysis, notably at 750 nm, 875 nm, and 900 nm. Comparing broadband NIR and far-red light (750 nm) showed no significant difference in hemolysis reduction. Pulse-dosed NIR irradiation allowed safe increases in radiation dose, effectively limiting hemolysis at higher doses where continuous exposure was harmful. These findings highlight NIR photobiomodulation's potential in protecting RBCs from oxidative stress and will be helpful in the effective design of novel medical therapeutic devices.

Near-infrared photobiomodulation of blood reversibly inhibits platelet reactivity and reduces hemolysis.

Photobiomodulation (PBM) in the red/near-infrared (R/NIR) spectral range has become widely recognized due to its anti-inflammatory and cytoprotective potential. We aimed to assess the effects of blood PBM on platelets function and hemolysis in an in vitro setting. Porcine blood samples were separated into four aliquots for this study, one of which served as a control, while the other three were subjected to three different NIR PBM dosages. The platelet count and functions and the plasma free haemoglobin and osmotic fragility of red blood cells were measured during the experiment. The control group had a considerable drop in platelet number, but the NIR exposed samples had more minimal and strictly dose-dependent alterations. These modifications were consistent with ADP and collagen-induced platelet aggregation. Furthermore, red blood cells that had received PBM were more resistant to osmotic stress and less prone to hemolysis, as seen by a slightly lower quantity of plasma free hemoglobin. Here we showed under well-controlled in vitro conditions that PBM reversibly inhibits platelet activation in a dose-dependent manner and reduces hemolysis.

The effect of red-to-near-infrared (R/NIR) irradiation on inflammatory processes.

Introduction: Near-infrared (NIR) and red-to-near-infrared (R/NIR) radiation are increasingly applied for therapeutic use. R/NIR-employing therapies aim to stimulate healing, prevent tissue necrosis, increase mitochondrial function, and improve blood flow and tissue oxygenation. The wide range of applications of this radiation raises questions concerning the effects of R/NIR on the immune system. Methods: In this review, we discuss the potential effects of exposure to R/NIR light on immune cells in the context of physical parameters of light. Discussion: The effects that R/NIR may induce in immune cells typically involve the production of reactive oxygen species (ROS), nitrogen oxide (NO), or interleukins. Production of ROS after exposure to R/NIR can either be inhibited or to some extent increased, which suggests that detailed conditions of experiments, such as the spectrum of radiation, irradiance, exposure time, determine the outcome of the treatment. However, a wide range of immune cell studies have demonstrated that exposure to R/NIR most often has an anti-inflammatory effect. Finally, photobiomodulation molecular mechanism with particular attention to the role of interfacial water structure changes for cell physiology and regulation of the inflammatory process was described. Conclusions: Optimization of light parameters allows R/NIR to act as an anti-inflammatory agent in a wide range of medical applications.

Applicability of FTIR-ATR Method to Measure Carbonyls in Blood Plasma after Physical and Mental Stress.

INTRODUCTION: Oxidative stress is a state of imbalance between the production of reactive oxygen species and antioxidant defenses. It results in the oxidation of all cellular elements and, to a large extent, proteins, causing inter alia the formation of carbonyl groups in their structures. The study focused on assessment of changes in the plasma protein-bound carbonyls in police horses after combat training and after rest and the applicability of infrared spectroscopy with a Fourier transform, utilizing the attenuated total reflectance (FTIR-ATR) in detecting plasma protein oxidation. METHODS: We evaluated the influence of both the different concentrations of hydrogen peroxide and combat training on protein carbonylation in horse blood plasma. The oxidation of plasma proteins was assessed using a spectrophotometric method based on the carbonyl groups derivatization with 2,4-dinitrophenylhydrazine (DNPH). The measured values were correlated with the carbonyl groups concentrations determined by means of the FTIR-ATR method. RESULTS: The linear correlation between the DNPH and FTIR-ATR methods was shown. The concentration of plasma protein-bound carbonyls significantly deceased in police horses after one-day rest when compared to the values measured directly after the combat training (a drop by 23%, p<0.05 and 29%, p<0.01 measured by DNPH and FTIR-ATR methods, respectively). These results were consistent with the proteins phosphorylation analysis. CONCLUSION: The FTIR-ATR method may be applied to measure the level of plasma proteins peroxidation.

Publisher Correction: Low-level light therapy reduces platelet destruction during extracorporeal circulation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Low-level light therapy reduces platelet destruction during extracorporeal circulation.

Extracorporeal circulation causes many deleterious effects on blood cells. Low-level light therapy (LLLT) in the red/near-infrared spectral range is known for its cytoprotective properties but its use during cardiopulmonary bypass (CPB) has not yet been studied. We aimed to assess whether LLLT protects platelets during CPB. 24 pigs were connected to 1-hour-CPB and observed for the next 23 hours. In 12 animals, blood circulating through the oxygenator was treated with LLLT. Platelet count and function were monitored throughout the experiment. The decrease in platelet count was greater in the control group, especially during CPB and after 24 hours. In LLLT group CD62P expression remained quite stable up to the 12th hour of the experiment, whereas in the control group it continuously decreased till the end of observation. Platelets in the control group were more prone to aggregation in the postoperative period than at the beginning of the experiment, whereas platelets in the LLLT group aggregated similarly or less intense. Limitation of platelet loss, pattern of aggregation and CD62P expression suggest that LLLT may stabilize platelet function during CPB and diminish the negative effects associated with the interaction of cells with an artificial surface.

Low-Level Light Therapy Protects Red Blood Cells Against Oxidative Stress and Hemolysis During Extracorporeal Circulation.

Aim: An activation of non-specific inflammatory response, coagulation disorder, and blood morphotic elements damage are the main side effects of the extracorporeal circulation (ECC). Red-to-near-infrared radiation (R/NIR) is thought to be capable of stabilizing red blood cell (RBC) membrane through increasing its resistance to destructive factors. We focused on the development of a method using low-level light therapy (LLLT) in the spectral range of R/NIR which could reduce blood trauma caused by the heart-lung machine during surgery. Methods: R/NIR emitter was adjusted in terms of geometry and optics to ECC circuit. The method of extracorporeal blood photobiomodulation was tested during in vivo experiments in an animal, porcine model (1 h of ECC plus 23 h of animal observation). A total of 24 sows weighing 90-100 kg were divided into two equal groups: control one and LLLT. Blood samples were taken during the experiment to determine changes in blood morphology [RBC and white blood cell (WBC) counts, hemoglobin (Hgb)], indicators of hemolysis [plasma-free hemoglobin (PFHgb), serum bilirubin concentration, serum lactate dehydrogenase (LDH) activity], and oxidative stress markers [thiobarbituric acid reactive substances (TBARS) concentration, total antioxidant capacity (TAC)]. Results: In the control group, a rapid systemic decrease in WBC count during ECC was accompanied by a significant increase in RBC membrane lipids peroxidation, while in the LLLT group the number of WBC and TBARS concentration both remained relatively constant, indicating limitation of the inflammatory process. These results were consistent with the change in the hemolysis markers like PFHgb, LDH, and serum bilirubin concentration, which were significantly reduced in LLLT group. No differences in TAC, RBC count, and Hgb concentration were detected. Conclusion: We presented the applicability of the LLLT with R/NIR radiation to blood trauma reduction during ECC.

Effects of Near Infrared Radiation on DNA. DLS and ATR-FTIR Study.

We presume that the primary effect of Near Infrared (NIR) radiation on aqueous solutions of biological molecules concerns modification of hydrogen bonded structures mainly the global and the hydration shell water molecules. Since water has a significant influence on the DNA structure, we expect that the thermal stability of DNA could be modified by NIR radiation. The herring sperm DNA was exposed to NIR radiation (700-1100nm) for 5, 10, and 20min periods. The temperature dependent infrared measurements were done for the thin films formed on the diamond ATR crystal from evaporated DNA solutions exposed and unexposed to NIR radiation. For the NIR-treated samples (at room temperature) the B form was better conserved than in the control sample independently of the irradiation period. Above 50°C a considerable increase in the A form was only observed for 10min NIR exposed samples. The hydrodynamic radius, (Rh), studied by the dynamic light scattering, showed drastic decrease with the increasing irradiation time. Principal components analysis (PCA) allowed to detect the spectral features correlated with the NIR effect and thermal stability of the DNA films. Obtained results strongly support the idea that the photoionization of water by NIR radiation in presence of DNA molecules is the main factor influencing on its physicochemical properties.

Sheep model of haemodialysis treatment.

More than two million patients received haemodialysis (HD) in 2013. Many methods for improving dialysis therapy outcomes have been tested. Nevertheless, patients continue to experience high morbidity and mortality rates. We aimed to develop an animal model of HD treatment to study methods that would prevent the adverse effects of renal replacement therapy. The study was conducted using six male Merino sheep. The animals underwent a two-step bilateral nephrectomy, and a permanent dual-lumen catheter was inserted into the jugular vein. In each animal, 10 short, daily HD treatments were conducted. The dialysis prescription was adjusted individually to each animal. Measures of dialysis adequacy (spKt/V and urea reduction ratio [URR]) were calculated for each HD treatment. All animals remained in a good clinical state during the experiment. However, a sustained decrease in red blood cell count was detected. The average URR was 0.65 ± 0.01, whereas the calculated spKt/V was approximately 1.16 ± 0.03. Neither hyperphosphataemia nor a significant decline in serum albumin concentrations were detected during the study. A sustained increase in serum potassium concentrations was detected on consecutive days of the experiment. All sheep survived the treatment and were euthanized at the end of the experiment. In conclusion, we developed a reproducible sheep model of HD treatment. The gentle nature and specific anatomical features of sheep provided easy blood access and allowed us to perform HD without pharmacological intervention. However, some differences in sheep physiology relative to human physiology must be considered when interpreting the results of the study.

Comparison of FTIR-ATR and Raman spectroscopy in determination of VLDL triglycerides in blood serum with PLS regression.

Hypertriglyceridemia, related with triglyceride (TG) in plasma above 1.7mmol/L is one of the cardiovascular risk factors. Very low density lipoproteins (VLDL) are the main TG carriers. Despite being time consuming, demanding well-qualified staff and expensive instrumentation, ultracentrifugation technique still remains the gold standard for the VLDL isolation. Therefore faster and simpler method of VLDL-TG determination is needed. Vibrational spectroscopy, including FT-IR and Raman, is widely used technique in lipid and protein research. The aim of this study was assessment of Raman and FT-IR spectroscopy in determination of VLDL-TG directly in serum with the isolation step omitted. TG concentration in serum and in ultracentrifugated VLDL fractions from 32 patients were measured with reference colorimetric method. FT-IR and Raman spectra of VLDL and serum samples were acquired. Partial least square (PLS) regression was used for calibration and leave-one-out cross validation. Our results confirmed possibility of reagent-free determination of VLDL-TG directly in serum with both Raman and FT-IR spectroscopy. Quantitative VLDL testing by FT-IR and/or Raman spectroscopy applied directly to maternal serum seems to be promising screening test to identify women with increased risk of adverse pregnancy outcomes and patient friendly method of choice based on ease of performance, accuracy and efficiency.

Near infrared light induces post-translational modifications of human red blood cell proteins.

There is a growing body of evidence that near infrared (NIR) light exerts beneficial effects on cells. Its usefulness in the treatment of cancer, acute brain injuries, strokes and neurodegenerative disorders has been proposed. The mechanism of the NIR action is probably of photochemical nature, however it is not fully understood. Here, using a relatively simple biological model, human red blood cells (RBCs), and a polychromatic non-polarized light source, we investigate the impact of NIR radiation on the oxygen carrier, hemoglobin (Hb), and anion exchanger (AE1, Band 3). The exposure of intact RBCs to NIR light causes quaternary transitions in Hb, dehydration of proteins and decreases the amount of physiologically inactive methemoglobin, as detected by Raman spectroscopy. These effects are accompanied by a lowering of the intracellular pH (pHi) and changes in the cell membrane topography, as documented by atomic force microscopy (AFM). All those changes are in line with our previous studies where alterations of the membrane fluidity and membrane potential were attributed to NIR action on RBCs. The rate of the above listed changes depends strictly on the dose of NIR light that the cells receive, nonetheless it should not be considered as a thermal effect.

Application of FTIR-ATR Spectroscopy to Determine the Extent of Lipid Peroxidation in Plasma during Haemodialysis.

During a haemodialysis (HD), because of the contact of blood with the surface of the dialyser, the immune system becomes activated and reactive oxygen species (ROS) are released into plasma. Particularly exposed to the ROS are lipids and proteins contained in plasma, which undergo peroxidation. The main breakdown product of oxidized lipids is the malondialdehyde (MDA). A common method for measuring the concentration of MDA is a thiobarbituric acid reactive substances (TBARS) method. Despite the formation of MDA in plasma during HD, its concentration decreases because it is removed from the blood in the dialyser. Therefore, this research proposes the Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) spectroscopy, which enables determination of primary peroxidation products. We examined the influence of the amount of hydrogen peroxide added to lipid suspension that was earlier extracted from plasma specimen on lipid peroxidation with use of TBARS and FTIR-ATR methods. Linear correlation between these methods was shown. The proposed method was effective during the evaluation of changes in the extent of lipid peroxidation in plasma during a haemodialysis in sheep. A measurement using the FTIR-ATR showed an increase in plasma lipid peroxidation after 15 and 240 minutes of treatment, while the TBARS concentration was respectively lower.

Individual osmotic fragility distribution: a new parameter for determination of the osmotic properties of human red blood cells.

The aim of our experiments was to characterise and to validate the osmotic fragility test when applied to human blood samples with no significant alterations of osmotic fragility but with a differentiating shape of the haemolysis curve. All experiments were carried out on human erythrocytes taken from the Regional Centre of Blood Donation and Blood Therapy in Wroclaw. The washed erythrocytes were exposed to near-infrared radiation (NIR) or ozonated, and the osmotic fragility test was applied. The osmotic fragility, calculated from the experimental haemolysis curve for the control and cells irradiated for 15 min, is the same within the empirical error. Calculation of the first derivative of the haemolysis curve allowed us to visualise the changes in osmotic fragility distribution after exposure to NIR. By contrast, significant changes both to the osmotic fragility value and the distribution of osmotic properties were observed after an erythrocytes ozonation procedure. Description of cell osmotic properties requires at least two parameters-the value of osmotic fragility and the slope of the haemolysis curve in the region where absorbance sharply increases due to cell haemolysis.

[Are disinfectant residues remained after cleaning hemodialysis machine procedure safe for patients?]. / Czy sladowe stezenia srodków dezynfekujqcych sztuczna nerke sa bezpieczne dla pacjentów hemodializowanych?

The dialysis machine shall be cleaned and disinfected after each patient treatment or after every 72 hours break in working. An acceptable disinfectants such as Puristeril plus or Puristeril 340, Citrosteril, Diasteril and Sporotal are used for decontamination. Puristeril 340 is designed for cold disinfection and due to the low pH value, the necessary decalcification of hemodialysis machines is easily achieved. It can be used for all haemodialysis systems like hemodialysis machines, water treatment devices and circuit pipes. Diluted Puristeril decomposes in a non-toxic way. Degradation products of peracetic acid, which is main component of Puristeril are: hydrogen peroxide and acetic acid. Peracetic acid is widely used for disinfection due to its exceptionally broad spectrum of microbiocidal activity at low concentrations and short exposure times. After use Puristeril is easily removable by rinsing with water. This paper deals with the effect of the Puristeril toxicity on blood as a function of its concentration and incubation time. Concentration range of 3.5-70 ppm was used, with particular emphasis on concentrations close to 5 ppm, a value is the limit of sensitivity of strips of starch potassium iodide, the tests for detection of peracetic acid. There was a strong increase in autohaemolysis and malondialdehyde concentrations with increasing concentration of Puristeril. There were also changes in dependence on the parameters of the incubation time, with the greatest effects obtained after 2 hours incubation with Puristeril. The detection limit of peracetic acid used strips of starch potassium iodide does not guarantee the safety of a patient undergoing hemodialysis. Even the residual concentration of Puristeril plus cause increased lipid peroxidation of membrane, and therefore suggest the routine use of stripes on the lower limit of detection of peracetic acid or implement measurement of hydrogen peroxide residues performed with sensitivity 1 ppm.

Spectroscopic techniques in the study of human tissues and their components. Part I: IR spectroscopy.

Among the currently used methods of monitoring human tissues and their components many types of research are distinguished. These include spectroscopic techniques. The advantage of these techniques is the small amount of sample required, the rapid process of recording the spectra, and most importantly in the case of biological samples - preparation of tissues is not required. In this work, vibrational spectroscopy: ATR-FTIR and Raman spectroscopy will be used. Studies are carried out on tissues: tendons, blood vessels, skin, red blood cells and biological components: amino acids, proteins, DNA, plasma, and deposits.

Spectroscopic techniques in the study of human tissues and their components. Part II: Raman spectroscopy.

Among the currently used methods of monitoring human tissues and their components many types of research are distinguished. These include spectroscopic techniques. The advantage of these techniques is the small amount of sample required the rapid process of recording the spectra, and most importantly in the case of biological samples - preparation of tissues is not required. In this work vibrational spectroscopy: ATR-FTIR and Raman spectroscopy will be used. Studies are carried out on tissues: tendons, blood vessels, skin, red blood cells and biological components: amino acids, proteins, DNA, plasma, and deposits.

Structural alteration of collagen fibres--spectroscopic and mechanical studies.

Fourier Transform Near Infrared Raman Spectroscopy has been used to monitor the molecular changes of collagen in a tendon subjected to strain. In the Raman spectrum of the unstrained tendon, some protein bands, mainly assigned to collagen, can be observed: amide I (1666 cm-1) and III (1266 and 1248 cm-1) vibrational modes and skeletal (C-C) stretching vibrations (816 and 940 cm-1). The position of these bands is changing with the increasing strain values. It is concluded that elastin and non-helical domains of collagen are initially involved in the load transfer and triple helices of collagen are gradually joining this process.

Usefulness of spectroscopy for biomedical engineering.

Modifications of phenylalanine amino acid after its exposure to near-infrared (NIR) radiation have been investigated using ATRFTIR (Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy). The process of amino acid aggregation after its exposure to NIR has been observed. A possible mechanism of amino acid dimer formation has been proposed with the help of theoretical calculations of quantum mechanics (MP2 and B3LYP/6-31 G* level) using the GAUSSIAN 03 package. The usefulness of spectroscopy for biomedical engineering is discussed. ATR-FTIR appears to be a powerful tool for measuring tissue damage in aqueous environments.

Influence of silybin on biophysical properties of phospholipid bilayers.

AIM: Silybin (silibinin) is major biologically active flavonolignan extracted from milk thistle (Sylibum marianum). Its biological activities include hepato-protection, anticancer properties, and antioxidant- and membrane-stabilizing functions. Although membranes are postulated to be one of the cellular targets for silybin, little is known about its interaction with phospholipid bilayers. METHODS: In the present work, the interactions of silybin with phosphatidylcholine bilayers were studied in detail using fluorescence spectroscopy, microcalorimetry and electron spin resonance techniques. RESULTS: The results showed that silybin interacted with the surface of lipid bilayers. It affected the generalized polarization of the fluorescent probe Prodan, while not influencing the more deeply located Laurdan. Silybin lowered the main phospholipid phase transition temperature as judged by microcalorimetry, and caused the immobilization of spin probe Tempo-palmitate located on the surface of membranes. The mobility of spin probes 5- and 16-doxyl stearic acid was not affected by silybin. Silybin-induced quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence indicated that some flavonoid molecules partitioned into the hydrophobic region of membranes, which did not change significantly the biophysical properties of the deeper membrane regions. CONCLUSION: Such a behavior of silybin in membranes is in accordance with its postulated biological functions and neglectable side effects of therapies using silybin.

[Does daily hemodialysis influence urea kinetic modeling (UKM) coefficients?--Preliminary report]. / Czy hemodializa codzienna wplywa na zmiane wspólczynników modelowania kinetycznego dializy?--Doniesienie wstepne.

Large number of data shows beneficial effects of implementing daily hemodialysis (DH) upon the outcome in patients dialysed previously in 3 times a week hemodialysis (3H) schedule. The mechanisms responsible for this phenomenon are still unclear, despite the time of low-flux DH sessions is shortened almost by half. Evaluation of the effect of doubling the number of hemodialyses per week upon so called cellular clearance (intercompartmental diffusion coefficient, Kc) computed in 2 pool-model was main aim of this study. 6 chronically dialysed patients (previously 3x per week) were subjected to DH. Based upon output data from UKM and weekly KT/V, the time for each DH session was computed, with no change in Kd (dialyser clearance). Kc was estimated from double-pool volume variable model equations and rebound. By the use of almost similar dialyser clearances in 16 conventional and 29 DH modeling sessions, estimated Kc values had been found non significantly higher in DH: (323.16; S.D. 187.86 vs. 268.80; S.D. 104.09 ml/min; p=0,68). Mean ultrafiltration/pre-dialysis body weight ratio (UFR/BW1) was 4,97 (S.D. 2.27)% in conventional hemodialysis and 3.66 (S.D. 1.46)% in DH. Mean dialysis index Kt/V values had decreased in DH (0.79; S.D. 0.17, vs. 1,34 (S.D. 0.26). Mean UFR/W1 ratio correlated negatively with Kc either in conventional or in DH (r=-0.653; p = 0.006 and r=-0.552; p=0.0036, respectively). Statistically significant negative correlation between Kt/V and Kc was found only in DH subjects (r=-0.466, p =0.010). The authors concluded, that increased Kc observed in patients subjected to DH may be responsible for better dialysis efficacy in patients switched into this treatment modality.