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Sialic Acid Content of the Erythrocyte and of an Ascites Tumor Cell of the Mouse AARON MILLER, JQHN F. SULLIVAN, AND JAY H. KATZ (P.AJ4ioisotope and Medical Services of the Boston Veterans Adminjairation Hospital, and the Department of Medicine, Boston University @SchoolofMedicine, Boston, Massachusetts) SUMMARY The sialic acid content of the mouse erythrocyte was compared with that of the Ehrlich ascites carcinoma cell. Neuraminidase treatment of intact tumor cells yielded 36 times more sialic acid than was released from the erythrocyte on a per cell basis. If this were all located on the surface, it may be calculated that the density of sialic acid on the Ehrlich ascites carcinoma cell is about 4 times greater than that on the erythro cyte. Trypsin released a sialic acid-containing fragment from both types of cells, the sialic acid being in the “bound―form.The sialic acid released by trypsin treatment of Ehrlich ascites tumor cells was only about one-sixth of that released by neuraminidase. By contrast, approximately equal amounts were liberated from the erythrocytes by these two enzymes. The erythrocyte sialic acid-containing substance was nondialyzable and was not precipitated by trichioroacetic acid. N-acetylneuraminic acid constituted approximately 70 per cent and N-glycolyneuraminic acid the remaining 30 per cent of the sialic acid of the tumor cell as determined by paper chromatography. In the erythrocyte N-acetylneuraminic acid constituted almost all the sialic acid, N-glycolyl neuraminic acid being undetectable in this cell. The sialic acids are a family of substances ured isoelectric point of about pH 4.0 (18). It formed by the condensation of N- or 0- substitut seemed possible that sialic acid might be respon ed mannosamine with phosphoenol pyruvate sible for the negative charge of the EAT cell. In (17, 923). They are strongly acidic compounds this study the concentration of sialic acid in the whose terminal carboxyl group has a pK of about mouse EAT cell was determined and compared 92.6 (19). Sialic acids have been found on the eryth with that of the mouse erythrocyte. While this rocyte membranes of a number of vertebrate investigation was under way, Wallach and Eylar species (5, 7, 192). Viral or bacterial neuramini reported on the presence of sialic acid in the mouse dases, enzymes which cleave the a-ketosidic link EAT cell (921).The present study corroborates age between a terminal sialic acid group and a their findings and presents further data relating sugar or sugar derivative (9), can liberate free to the subject. sialic acid from such red cell membranes (5, 7). The surface of the mammalian erythrocyte is neg MATERIALS AND METHODS atively charged with a reported isoelectric point Blood was obtained by cardiac puncture from of about pH 92.0 (1, 8). Following enzymatic re 6- to 8-month-old female Swiss mice. Heparin was moval of sialic acid from the human erythrocyte, used as anticoagulant. Blood was pooled from a the isoelectric point of the red cell is greatly in number of animals (up to fifteen), depending on creased, indicating that these groups are primarily the amount of blood needed. The blood was centri responsible for the negative charge on the erythro fuged at 1,400 X g for 920minutes at 4°C., after cyte membrane (5, 7). which the plasma and buffy coat were removed. The mouse Ehrlich ascites tumor (EAT) cell The erythrocytes were washed 3 times with cold also has a negatively charged surface with a meas 0.15 M NaCl and then made up to approximately Receivedfor publication October 19, 1962. the original volume. When “ghosts―were to be 485 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. 486 Cancer Research \@ol @3,March 1963 prepared, the washed cells were centrifuged again 370 C. in a Dubnoff shaker for 1 hour. Following at 1,700 X g for 45 minutes, and the supernatant incubation, the contents were centrifuged, and the fluid was removed. The ghosts were prepared from supernatants were stored at 4°C. until the con 1- or 92-mi. aliquots of the packed red cells by the centration of sialic acid was determined. All exper method of Tishkoff et al. (920). Tumor cells were iments were carried out on triplicate aliquots of obtained 7—8days after the intraperitoneal injec cells. tion of 3-month-old female Swiss mice with 0.3 ml. The total sialic acid in mouse red cells was deter of a suspension of a hyperdiploid EAT. The tumor mined on aliquots of ghosts equivalent to 1 or 92 had been previously propagated at 7-day intervals. ml. of packed erythrocytes. Three ml. of 0.1 N Any ascitic fluid grossly contaminated with red H2SO4was added to 1 ml. of ghosts, and the con cells was discarded. The EAT cells were centrifuged tents were then hydrolyzed at 80°—85°C.for 1 at 100 X g at 4°C., and the supernatant plas hour. After hydrolysis the ghosts were centrifuged, ma and the few red cells at the top of the EAT cell and the supernatant, together with two 3-mi. 0.1 column were removed and discarded. The cells N H2SO4 washings, were saved for sialic acid meas were then washed S times with 4 volumes of urement. In preliminary studies it was found that Krebs-Ringer bicarbonate, pH 7.4, containing 11 1 hour of hydrolysis was not sufficient for maxi mmoles glucose. At the end of the last wash the mum recovery of red cell sialic acid. Therefore, a cells were returned to their original volume with second 1-hour period of hydrolysis was routinely buffer. performed, the supernatants of both hydrolysis Sialic acid determination.—Sialic acid was periods being combined for sialic acid determina measured by the method of Warren (9292),except tion. that 0.5-nd. aliquots of sample were used with a EAT cell homogenates were prepared by freeze commensurate doubling of the volume of periodate thawing whole cells S times. The measurement of added. The reaction product followed Beer-Lam total sialic acid on these homogenates by acid hy bert's law within the limits defined by Warren. drolysis was not feasible, owing to the presence of Crystalline N-acetyl neuraminic acid' (NANA) interfering substances in the sialic acid determina was used as a reference standard with each sialic tions. This problem was also noted by Wallach assay. No correction was made for the N-glycolyl and Eylar (921). The interfering substances could neuraminic acid in EAT cells or for the trace not be eliminated by resin column chromatogra amount of an unknown sialic acid found in eryth phy on Dowex 50W-X8 and Dowex 92-X8 (19) or rocytes. by extraction with a number of organic solvents. In a typical experiment (a) total sialic acid, The use of the optical density readings at 5392and (b) sialic acid released by neuraminidase, and 5692 mjs instead of the 5392 and 549 ms readings as (c) sialic acid released by trypsin were determined. suggested by Warren (@92)and adopted by Wallach All glassware was siliconized. One-half ml. of the (921) did not materially improve the accuracy of packed erythrocytes and 1 ml. of EAT cell suspen the results. When cell homogenates were incubated sion were pipetted into a 50-mi. Erlenmeyer flask. with neuraminidase at 37°C. for 1—92hours,mate The red cell pipette was rinsed with three 0.5-mi. rials absorbing light at 5392m@ were not released. aliquots of 0.15 M NaCl to insure quantitative de Therefore, only the sialic acid released by neura livery of the packed erythrocytes. Neuraminidase minidase treatment of cell homogenates@could be prepared from Cholera vibrio2 (0.5- to 3.0-ml. ali measured. For the determination, homogenates quots) or trypsin8 three times recrystallized (1—8 from I ml. of EAT cells were mixed with 1 ml. of mg.), dissolved in 1 ml. of Krebs-Ringer phos Krebs-bicarbonate buffer pH 7.4 and 1 ml. of the phate, pH 7.6, was added to cell suspensions. Con neuraminidase solution. After incubation for 1 troi flasks without added enzyme were included in hour at 37°C., the mixture was centrifuged, and each experiment. The cells were incubated at the free sialic acid was determined on the super natant. In the estimation of total volume of super 1 Obtained from Dr. E. Eylar. Additional material obtained from General Biochemicals Inc. was also found to be satisfac natant, 80 per cent of the packed EAT cell mass tory. was assumed to be due to intracellular water. 2 Behringwerke, West Germany. Enzyme was dissolved in Duplicate 0.1-mi. aliquots of packed red cells 0.15 M NaC1 solution containing 9 m@sCaCl, and 0.05 M Na were diluted with 100 ml. of isotonic saline. The acetate buffer, pH 5.5. One ml. of the enzyme preparation con EAT cells were diluted with S per cent acetic acid. tamed 100 units of enzyme activity, one unit being equal to the Both were counted in a hemacytometer. Hemato amount of enzyme that would liberate 1 @g.ofsialic acid from a glycoprotein substrate during a 15-minute incubation period crits and tumor cell volumes were measured in at 370 C. duplicate. No correction was made for the volume 3 Nutritional Biochemicals and Sigma Chemical Co. of trapped fluid in either of the cell types. The cal Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. MILLER et al.—Tumor Cell and Erythrocyte S'ialic Acid 487 culated mean corpuscular volumes (MCV) were (b) sec-butanol-acetone-acetic acid-water (6:6: @ found to be 54 cu.

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