Autoradiography of Diffusible Compounds in Human Nonnucleated Erythrocytes: Studies with Tritiated Glucose and Adenine

A simple method for autoradiography of diffusible substances in erythrocytes is described. It is based on retarding diffusion from the red corpuscles during fixation and processing by using the bifunctional aldehyde, glutaraldehyde; rapid drying; and applying the nuclear emulsion as a dried, preformed film.

The technique was used for studying the changes during storage in the ability of red blood cells to utilize glucose. The incorporation of glucose-6-³H by samples of blood stored for different periods of time was followed by the number of silver grains present in the red corpuscles. It was observed that the percentage of erythrocytes with fewer than four grains increased progressively during the storage of ACD blood. The incorporation of glucose as observed by the silver grain pattern was reversible toward that of freshly collected red corpuscles following incubation of the stored samples with adenosine.

Similar studies were performed using adenine-8-³H as the label in blood stored for six weeks in ACD adenine solution. Incubation of the blood samples containing 0.5 mM. adenine for one hour at 37°C resulted in autoradiographic patterns equivalent in grain counts to those given by red blood cells stored with the radiolabeled adenine for five weeks. The incubation of fresh samples with inosine was found to inhibit the incorporation of adenine, whereas inosine enhanced the number of grains in red blood cells which had "run down" during storage.

The autoradiographic technique presented should be of value for the in vitro evaluation of methods of blood preservation and for studying the metabolic heterogeneity of normal red corpuscles and other tissue cells. It should be useful for investigating hereditary and acquired abnormalities of metabolism.