As most of these changes did not occur with the red cells in suspension in different isotonic saline solutions, we have attempted to explore the relationship of plasma to red cells and lipids. This was done in the following manner. The cholesterol content of red cells was seen to be progressively lowered by repeated washings with isotonic saline. When the amount of cholesterol is reduced below a certain level, hemolysis ensues. Standardizing these washings by replacing the plasma with an equal amount of saline, hemolysis is usually obtained in some bloods after 1 or 2 washings while in most after more than 10 washings. This occurs when the cholesterol content of the red cells falls below 58 mgm. %. When the separated plasma is again added to these repeatedly washed and consequently cholesterol impoverished blood cells, their cholesterol rises. (Table XXXI) By further additions of new portions of the same plasma, the cholesterol content of the red cells can be progressively elevated to the original values. Apparently cholesterol passes readily from the plasma to the red cells. This was confirmed by measuring the cholesterol content of the plasma before and after it was mixed with cholesterol impoverished cells. After treating a portion of citrated plasma several times with other portions of cholesterol impoverished red cells, the amount of cholesterol in the plasma was markedly reduced. The addition of the unwashed red cells to this cholesterol impoverished plasma raised back its cholesterol content. The content could be increased still further almost back to the previous values by repeating this procedure. (Table XXXII) It was clear that a balance in the cholesterol content seems to be realized between plasma and red cells through possible passages in both directions. This fact would imply that the red cells may serve as a buffer reserve for rapid changes occurring in the plasma sterols.

The treatment of red cells in vitro

(a)

The treatment of red cells in vitro

(b)

Fig. 264. The treatment of red cells in vitro with conjugated fatty acids (trienes) induces the appearance of vacuoles, (a) In spontaneous lesions characterized by an off balance type D with a predominance of fatty acids or in lesions induced by the administration of conjugated trienic fatty acids, vacuolated red cells are seen, (b).

Table XXXI. Influence Of Citrated Plasma Upon Red Cell Cholesterol

Before Treatment

186 mgr.%

After 10 Washings

62 "

After First Treatment

with Plasma

111

After Second Treatment

with Plasma

142 "

The role of red cells in the transportation and distribution of fatty acids through the blood not only appears more evident, but also indicates a selective intervention. When plasma was treated as mentioned above with fatty acids that were easily identifiable, and then mixed with the red cells, an unequal distribution between plasma and red cells was seen. The different influence of the different fatty acids became obvious. Saturated fatty acids could not be found in the red cells when these acids were used, while unsaturated fatty acids seemed to be selectively retained. Fatty acids such as oleic, linolenic, eleostearic, or norbixine were found convenient to be used for this purpose. They were easily identified, the first through its chemical character, the second after conjugation, and spectral analysis, the third through its characteristic absorption in ultraviolet light, and the last through its color in chromatographic column. After mixing with the red cells, they were found unequally and selectively fixed to the red cells: least for oleic, fixation was found to increase with the degree of desaturation. The same fixation was seen to occur in vivo. When animals were treated with saturated fatty acids, these substances did not appear in the red cells. When treated with oleic, linolenic, eleostearic acid or norbixine, the content of these acids in the red cells was found to be for the last acids as much as five times higher than in plasma. A selective fixation of those fatty acids on the red cells could thus be recognized. This appeared still more striking when compared with that of cholesterol. In animals treated with cholesterol, the relative proportions between the proportion in plasma and cells was not seen to be altered by a total increase in cholesterol. It appears that the red cells have the capacity of selectively fixing from the plasma certain fatty acids, particularly the more unsaturated ones.

Table XXXII. Influence Of Red Cells Upon Plasma Cholesterol

Before Treatment

226 mgr.%

After Fifth Treatment with

Washed Red Cells

96 "

After First Addition of

Unwashed Red Cells

163 "

After Second Addition of

Unwashed Red Cells

180 "