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The presence of an erythropoietic factor in the plasma of animals subjected to hypoxic hypoxia has been reported recently by Stohlman( 1 ) and Prentice and Mirand( l a ) . Additional data from our laboratory bear out a time-concentration relationship of plasma erythropoietin (EPF) in hypoxic hypoxic rats. Knowing of this time-concentration of EPF, studies were carried out to determine whether this time-concentration relationship of plasma EPF can be altered in absence of the kidney (s) and/or the spleen. Jacobson et aZ. (2) reported recently that bleeding and cobalt chloride administration did not produce circulating erythropoietin in the absence of the kidneys and postulated that the kidney is the site of production of EPF. The purpose of this report is to demonstrate that hypoxic hypoxic rats with or without their kidney(s) and/or spleen can produce erythropoietin.Material and methods. Sprague-Dawley rats were placed in chambers constructed of plexiglass. Into each chamber was directed a mixture of nitrogen and oxygen, the proportions of which, along with ambient pressure, determined the simulated altitude. Flow of nitrogen and oxygen was regulated by a rotameter type of flow-meter and remained quite constant over the experimental period. The chamber 0 2 concentration was checked by reading of the oxygen and nitrogen flowmeters and also by a portable Beckman Oximeter. Random air samples were also taken from the chamber in a Douglas bag and checked with a mass spectrograph for 02, N2 and COS. Good agreement in results was obtained. A flow of approximately 5 liters/minute was maintained through the chamber and soda lime and sulfuric acid were placed in the chamber to minimize C02 and humidity levels.Sprague-Dawley rats, 2 to 3 months old, used as donors, were exposed in a chamber to an atmosphere of approximately 10% 0 2 for the time intervals shown in Table I. Rats of another group, used as donors, were nephrectomized and/or splenectomized and caution was taken during the operation that no bleeding occurred. They were then immediately placed in an atmosphere of 10% 0 2 a t various time intervals (Table 11). Food (Purina Laboratory chow) and H 2 0 were available to rats in the chamber at all times. At the end of the respective intervals the donor rats were withdrawn from the chamber and promptly exsanguinated from the dorsal aorta. A final hematocrit was done on each rat and no animal with hematocrit below 40 was used as a source of plasma. Plasma obtained was frozen and stored until used.Recipient animals were 3-month-old Sprague-Dawley rats which were used as assay animals approximately 15-20 days after hypophysectomy (3,4). They received 2 cc of donor rat plasma to be tested I.V. via the jugular vein on 2 successive days. On day 3 approximately 1 pc FeG9 was given I.V. via femoral vein and on day 4, a 24-hour FeJ9 uptake was done. The results for each group of animals were expressed as the average 24hour Fe59 uptake for the particular group.Results. The time-concen tra tion rela tionship of plasma EPF in hypo...

Rabbit red cells are separated by centrifuging them for one hour at 33,000 G in density gradient tubes of bovine serum albumin. The separation represented an equilibrium situation since rebanding experiments showed that the cells from a layer would again seek that density layer when recentrifuged in a new gradient tube. When rabbits were injected with 59Fe, the radioactive red cells at one day were nearly all light, but these labeled cells moved into the more dense layers over the next few days. This not only shows that the separation by density is discriminating but that some red cells became dense very quickly. Bearing in mind the problems of interpreting radioactive iron data because of the possibility of reutilization, it is tentatively concluded that dense red cells are not necessarily senescent red cells since these dense cells appear to persist for the normal life span of the rabbit red cell.

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