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 corpusclesduring fixation and processing by using the bifunctional aldehyde, glutaraldehyde; rapid drying; and applying the nuclear emulsion as a dried, preformedfilm.

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

Similar studies were performed using adenine-8-³H as the label in bloodstored for six weeks in ACD adenine solution. Incubation of the blood samplescontaining 0.5 mM. adenine for one hour at 37°C resulted in autoradiographicpatterns equivalent in grain counts to those given by red blood cells storedwith the radiolabeled adenine for five weeks. The incubation of fresh sampleswith inosine was found to inhibit the incorporation of adenine, whereas inosineenhanced 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 vitroevaluation of methods of blood preservation and for studying the metabolicheterogeneity of normal red corpuscles and other tissue cells. It should be useful for investigating hereditary and acquired abnormalities of metabolism.