Among the first experiments concerning the influence exerted by sterols and polyunsaturated fatty acids in vitro, were those concerned with the effects upon red cells. We have noted that when citrated blood is kept for two hours at 37°C in a test tube, the walls of which have been coated with crystals of cholesterol or with nonsaponifiable fractions obtained from various natural sources, the red cells become more swollen and turgescent, and less crenated than those not treated. Seen under the dark field microscope, the cell crown appeared uniformly more refringent. It was also noted that the treated red cells failed to form rouleaux or conglutinates similar to those seen as sludge in vivo. At the same time, the cells appeared richer in their sterol content. None of these changes were observed when the red cells were separated from their plasma and washed with saline and kept in a saline solution when treated with sterols, in the manner mentioned above.

Opposite effects were observed when fatty acids were added to blood. As the direct contact with the red cells produces hemolysis, the following technique was used. Fatty acid preparations especially as mixtures obtained from blood or cod liver oil, were added to heparinized or citrated plasma, thoroughly agitated and the excess separated by centrifugation. The plasma so treated was then added in various proportions to citrated or heparinized blood from the same subject. This portion of this blood was centrifuged and the treated plasma added to the supernatant plasma from which the same amount was withdrawn. The added plasma was mixed with the supernatant plasma and then this was mixed with the red cells. In this way hemolysis was prevented. Small amounts of treated plasma caused the red cells to shrink in size and frequently become crenated. In addition, a strong tendency to conglutinate which exceeded that noted in corresponding control specimens, was observed. When the quantity of fatty acid treated plasma exceeded a certain amount, hemolysis was induced. The addition of these two groups of lipids to red blood cells, have appeared to exert frank antagonistic effects.

* Delivered at Gordon Research Conferences, Kimball Academy, Meriden, New Hampshire. 1955

Sedimentation Rate

The two groups of lipids were also found to influence oppositely the red cell sedimentation rate in citrated blood. When citrated blood samples having high sedimentation velocities, were treated with cholesterol or an insaponifiable fraction in the manner described above, the speed of sedimentation was markedly reduced. Table XXVIII shows results obtained mentation rate tended to increase to abnormal values. This varied with the amount of treated plasma added. (Table XXIX) in different blood samples in which the sedimentation rate during one hour, was measured by the Westergren method. In general, it can be seen that the higher the sedimentation rate of the untreated sample, the greater was the effect of adding sterols.

On the other hand, when polyunsaturated fatty acids were added in the manner already described, to citrated blood from healthly subjects, the sedi-

Table XXVIII. Red Cell Sedimentation Rate (Mm./Hr.) Samples Treated With Unsaponifiable Fraction Of Blood Lipids

Control

Treated

110

12

96

19

81

18

48

15

18

10

12

6

9

7

8

8

6

5

Table XXIX. Red Cells Sedimentation RateóMm./Lst Hour. Quantity Of Treated Plasma Fatty Acid Used Control Added To 5 CC. Citrated Blood

1/4 cc.

1/2 cc.

1 cc.

Stearic

9

8

9

9

Palmitic

9

10

9

10

Linoleic

9

15

18

22

Linolenic

9

15

21

25

Cod Liver Oil

9

20

36

Hemolysis

Red Cell Volume

The same opposite effects of sterols and fatty acids were further observed upon the volume of red cells, as determined by the hematocrit, or also when the sedimentation in tubes was observed over a 24 hour period. (Table XXX) Sterol treated blood showed a significant increase in red cell volume, while on the other hand, with the addition of polyunsaturated fatty acids, the red cell volume decreased. This agrees with a retention of water by the cells in general when richer in sterols, which Schaeffer has described as the lipocytic index.

Table XXX. Changes In Volume Of Red Cells In Citrated Blood Treated In Vitro (Sedimentation After 24 Hours)

Substance Used

Control

Treated

Unsaponifiable fraction of blood

53

66

Stearic Acid

53

54

Saponifiable fraction of blood

53

50

On further analysis, these effects of fatty acids upon red cells mentioned above could be related to the polyunsaturation of these acids, since by treating the blood under the same conditions with saturated members, such as palmitic or stearic, these changes were not obtained.

The treatment of red cells with conjugated fatty acids, especially trienes. induces a marked vacuolization. This is seen to occur through an accumulation of part of the content of the red cell in droplets, strongly stained with eosin. (Fig. 264a) Similar changes are seen to occur in vivo. In lesions characterized by a predominance of fatty acids or induced by the administration of conjugated fatty acids, such vacuolated red cells are often seen. We used their presence, together with other characters, for the pathological diagnosis of the type D present in a lesion. (Fig. 264b)