Temperature and oxygenation dependence of haemoglobin and hemocyanin relaxation times at 9.4T

Introduction Proton relaxation rates of blood are beneath field strength depending on physiological parameters like haematocrit, ion compositon, oxygen saturation, pH and on physical variables like temperature (1, 2). Knowledge of correct T1 and T2 values are of considerable importance for quantification of fMRI signals, blood flow and perfusion in MR experiments (3, 4, 5) as well as for the quantification of tissue oxygenation levels in determining correct oxy/desoxy haemoglobin ratios (3). Furthermore, analysis of the proton relaxation times of water in the blood will give additional important physiological information and further insights into the bioenergetics of organisms (1). Since MRI and NMR studies using ectothermic animals as model organisms are becoming more and more frequent (e.g. 6), correct T1 and T2 values under these temperature conditions (in a temperature range of 0°C-20°C) are crucial for quantitative measurements of flow and perfusion with MRI in these organisms. Recently, it was shown that cephalopods are ideal model organisms for MR imaging and spectroscopy studies in neurological research under patho-physiological conditions (7). The potential use of these organisms for functional MRI studies relying on BOLD contrast, information on the magnetic properties of the cephalopod blood pigment haemocyanin depending on the oxygenation state is indispensable. To address this question the aim of this study was to determine the T1 and T2 values of water proton signals from purified haemoglobin and haemocyanin solutions at different temperature and oxygenation states. Materials and methods For the NMR studies on haemoglobin, blood (around 1 mL/sample) was taken from the caudal vein of North Sea cod acclimated at 10°C (Gadus morhua, n=3). Heparin (57.8 mg/1mL blood) was added used to prevent coagulation. Haematocrit values were around 30 Vol. % in all samples. T1 and T2 NMR studies were conducted on whole blood samples as well as on purified haemoglobin. Samples were washed, lysed, and centrifuged. The lysate was then applied to a SEPHADEX column (Pharmacia biotech, Sweden). Haemocyanin samples were taken from cephalopod blood from the Atlantic (Sepia officinalis, at 15°C water temperature, Arcachon, France). Haemocyanin is an extracellular blood pigment, therefore the blood samples were only centrifuged and supernatants were taken directly for the NMR studies. Fully oxygenated blood pigment samples were equilibrated by aerating the samples on a stirrer for 30 minutes at least prior the NMR experiments. For the NMR measurements ca. 450 mL of blood were transferred into a 5 mm NMR tube under aeration and sealed immediately. Blood pigment samples were deoxygenated by adding a few grains of sodium dithionite into the NMR tube right after the first set of NMR experiments. Figures 1a+b shows examples of oxygenated and deoxygenated blood pigments prior NMR experimentation. The clearly visible colour differences indicate the oxygenation state of the blood pigments. Oxygenated haemoglobin is cherry red and turns to dark red when deoxygenated (Fig. 1a), whereas fully oxygenated haemocyanin ranges from yellow to blue (depending on the functional units of the haemocyanin protein) and becomes colourless when deoxygenated (Fig 1b). Figure 1: