2nd YRA MedTech Symposium, Young Researchers Academy �– MedTech in NRW jointly held with the IEEE Workshop & SENSORICA 2017

Hochschule Ruhr West, June 8-9, Mülheim a. d. Ruhr, Germany, 2017

Effects of nitric oxide (NO) and ATP on red blood cell phenotype and deformability Katharina Schlemmer, Dariusz Porst, Rasha Bassam, Gerhard Artmann, Ilya Digel (1) Laboratory for Cell- and Microbiology Institute for Bioengineering, FH Aachen University of Applied Sciences D-52428 Jülich, Germany

E-Mail: k-schlemmer@web.de Web: www.zmb.fh-aachen.de Abstract �– Red blood cells (RBCs) are the most common type of blood cells in vertebrates and function as the principal means of delivering oxygen to the metabolizing tissues [1]. Alterations in the RBCs structure as well as changes in the functioning of their proteins can occur due to binding with a broad class of low-molecular weight modifiers constantly or transiently present in erythrocytes and elsewhere in the blood [2]. Among such modifiers, adenosine- 5�’-triphosphate (ATP) is probably the most important intra-erythrocyte organic phosphate in vivo. In red blood cells, the concentration of ATP is in the range of 0.2-2.0 mM and any variations away from this range may induce a pH dependent tetramerization of deoxyHb in vertebrates [3]. Another important species is nitric oxide (NO), which is a highly reactive free radical and has been identified as a regulatory molecule in a number of cellular systems, including, but not limited to, the immune, nervous and cardiovascular systems [4]. According to recent studies, erythrocytes can release substances such as adenosine triphosphate and nitric oxide into the blood as a part of their physiological responses [5]. Although both nitric oxide (NO) and ATP apparently play a significant role in blood functions, their exact function in controlling the principal RBC (mechanical) properties and therefore the blood circulation parameters remains poorly studied. A better understanding of the deformation of the red blood cells and their response on the substances in the blood is important not only for basic research but also for clinical use. The aim of our study was to investigate the influence of nitric oxide and ATP on the appearance and mechanics of human RBCs using the Microscopic Photometric Monolayer (MPM) technique. The MPM technique provides a tool to measure red blood cell (RBC) stiffness (resistance to elongation) and relaxation time. It combines many of the advantages of flow channel studies of point-attached RBCs with the simplicity, sensitivity and accuracy of photometric light transmission measurement [6]. The principal idea of this method is that the flow-induced bending and curvature change of RBC membrane is associated with the increase of light transmission. The monochromatic light having a wavelength of 415 nm passes through the flow chamber and is measured on the other side by a photometer as voltage output. 415 nm correspond to the maximum absorption spectrum of hemoglobin. In the flow chamber, the incident light is refracted at the erythrocyte surface, which causes changes in the light intensity. This technique allows the study of the effects of physicochemical factors on the elongation and relaxation time of the same cells within an average of four to five thousand cells adhered as a monolayer to glass. The measuring instrument consisted of the following components: syringe pump, flow chamber, light microscope, photometer and control unit. A dense monolayer of point-attached RBCs was prepared at the bottom of a flow-chamber. A steady-state flow, with stepwise increases of flow rate, induced the RBC elongation. The light transmission perpendicular through the monolayer plane was measured photometrically. The attached erythrocytes were treated with adenosine triphosphate and nitric oxide and the changes in the shape were followed in the time course, including wash-in/wash-out kinetics. Following a sudden flow stoppage, the RBCs returned to their resting shape and the RBC relaxation time was measured. The stiffness-relaxation time product, V (in mPas), was calculated to provide an estimate of viscosity. Established photometric methods

2nd YRA MedTech Symposium, Young Researchers Academy �– MedTech in NRW jointly held with the IEEE Workshop & SENSORICA 2017

Hochschule Ruhr West, June 8-9, Mülheim a. d. Ruhr, Germany, 2017

sensing tiny changes of red blood cell morphology at rest (red blood cell shape) and at very low shear forces (red blood cell stiffness, red blood cell relaxation time) were applied as well. The derivative induced effects were detected in a time- and dose-dependent manner.

Fig.1: The experimental setup (left) and the principle of the MPM �–technique (right). The experiments showed differences in both viscosity and deformability of the RBCs treated with ATP, NO-donors and NOS-inhibitors as compared to the control group.

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