The Filtrability of the Components of Alexin

Abstract

MUIR AND BROWNING (1909) were the first to show that alexin is retained by the filter when fresh serum is passed through a Berkefeld candle. Several filtrations are generally required to remove all traces of the alexic activity, although when a fresh candle is employed for each filtration, the third or fourth filtrate is usually devoid of alexic power (Schmidt, 1914). It is not established whether or not the failure of later filtrates to haemolyse sensitised erythrocytes results from the removal by the filter of some one or of all of the known components of alexin from the serum. We have, therefore, studied the effect of Berkefeld filtration upon the alexic activity of fresh human serum with respect to the thermolabile elements, and the thermostable third and fourth components (Whitehead, Gordon and Wormall, 1925;Gordon, Whitehead and Wormall, 1926). EXPERIMENTALThe fresh human serum was passed through a series of twelve properly prepared Berkefeld filters', and the various filtrates were tested for the presence of the several components of alexin. Portions of the same fresh serum which were treated by heat (56°C. for j hour), by zymin2, and by ammonium hydroxide3 and which alone were entirely without haemolytic power (see Table II), were used to test for the presence of the thermolabile elements, the third component and the fourth component respectively. 1 The filters were washed thoroughly with hot, dilute sodium hydroxide followed by distilled water until the filtrate was no longer alkaline to phenolphthalein.2 Zymin was prepared in a manner similar to that described by Whitehead, Gordon and Wormall (1925). A cake of Fleischmann's yeast was shaken with 25 c.c. of absolute alcohol for i hour, then with a like amount of absolute ether for the same period. To the residue was added 25 c.c. of physiological sodium chloride solution and the mixture was boiled for J hour. The sediment obtained by centrifugation represented the zymin employed in this work. The serum was inactivated for the third component by adding an excess (25 per cent. by volume) of the zymin and incubating the mixture for 2 hours.3 Ammonium hydroxide of 1/6.5 normality was used to inactivate the fourth component of the fresh serum. Twenty-five per cent. by volume of the ammonia was added to the serum and the mixture incubated at 370 C. for 11 hours (Gordon, Whitehead and Wormall, 1926). After its pH was adjusted to about 7-5, such a serum was satisfactory to test for the presence of the fourth component.

Studies on Destruction of Red Blood Cells. Ii. Chronic Hemolytic Anemia With Paroxysmal Nocturnal Hemoglobinuria: Certain Immunological Aspects of the Hemolytic Mechanism With Special Reference to Serum Complement 1

“…In the following observations a direct study of the effect of decreasing the complement concentration was undertaken. The reactive components of complement were removed in part or completely from human serum by each of 3 procedures: absorption with sensitized or unsensitized influenza bacilli; standing at room temperature; or by filtration through Berkefeld or Seitz filters (17). The hemolytic activity of the treated serums was then tested on patient's erythrocytes and compared with the activity of an unmanipulated sample.…”

“…been demonstrated by Strong and Culbertson (17) to remove all the known components of complement from fresh human serum. The thermolabile elements were the first to be removed and were not demonstrable in serum after from 3 to 6 filtrations.…”

Recent Advances in the Chemistry of Complement.

“…Its activity is destroyed by ultraviolet rays (33,84), although it is more resistant in this respect than midpiece and in the dry state is only slightly affected by ultraviolet light. Endpiece is readily adsorbed when passed through a Berkefeld filter (87).…”

“…C'4 is quite resistant to non-specific adsorption. Indeed, twelve consecutive filtrations through a Berkefeld filter are required to remove this component from serum (87). C'4 is far more resistant to ultraviolet light than are the thermolabile components of complement (33,84).…”