ABSTRACT - The transport of  C-amino acids into human erythrocytes and into erythrocytes which had been frozen and thawed was studied experimentally. The relative rates of uptake were found to be (in decreasing order): phenylalanine, leucine, alanine, trythophan, and glycine. Freezing (using gycerol as the cryoprotective additive) and thawing had no significant effect on either the magnitude of the rate constants or on the order of the above relative rates of amino acid uptake into erythrocytes suspended in isotonic TRIS-HCl buffer

Bizios, I. Rena

Moore, Richard

Parenti, Rita

English

University of Minnesota, Morris (UMM): Digital Well

Journal of the Minnesota Academy of Science Volume 37 Number 2 Number 2/3 Article 16 1971 Effect of Freezing and Thawing on Uptake of Amino Acids into Human Erythrocytes Richard Moore University of Minnesota Rita Parenti I. Rena Bizios Follow this and additional works at: https://digitalcommons.morris.umn.edu/jmas  Part of the Hematology Commons Recommended Citation Moore, R., Parenti, R., & Bizios, I. R. (1971). Effect of Freezing and Thawing on Uptake of Amino Acids into Human Erythrocytes. Journal of the Minnesota Academy of Science, Vol. 37 No.2, 119-121. Retrieved from https://digitalcommons.morris.umn.edu/jmas/vol37/iss2/16 This Article is brought to you for free and open access by the Journals at University of Minnesota Morris Digital Well. It has been accepted for inclusion in Journal of the Minnesota Academy of Science by an authorized editor of University of Minnesota Morris Digital Well. For more information, please contact skulann@morris.umn.edu. HEMATOLOGY Effect of Freezing and Thawing on Uptake of Amino Acids in.fo Human Erythrocytes DR. RICHARD MooRE*, DR. RITA PARENTI**, DR. I. RENA Bizms*** ABSTRACT - The transport of "C-amino acids into human erythrocytes and into erythrocytes which had been frozen and thawed was studied experimentally. The relative rates of uptake were found to be (in decreasing order): phenylalanine, leucine, alanine, trythophan, and glycine. Freez-ing (using gycerol as the cryoprotective additive) and thawing had no significant effect on either the magnitude of the rate constants or on the order of the above relative rates of amino acid uptake into erythrocytes suspended in isotonic TRIS-HCl buffer. The preservation of cells and tissues by freezing is be-coming a rp.ethod of increasing importance and frequent use in bacteriology, obstetrics, surgery, hematology, and animal husbandry. Various procedures and instruments have been proposed and/ or employed to process cells or tissues for long-term preservation. It is necessary and important, therefore, to evaluate these procedures and instruments. Freezing and thawing are assumed to affect the normal physiology of the red blood cells and to damage the cell membrane. Membrane transport studies provide a way for detecting and measuring the extent and the effect of such damages. In this respect, Wallach, et al., (1962) re-ported that frozen-deglycerolized human erythrocytes re-tain their normal cation flux and electrolyte concentra-tion. There are no results reported in connection with the amino acid uptake into frozen-thawed red cells. The transport of neutral amino acids into human erythro-cytes, however, has been the subject of numerous ex-perimental studies. A summary of the most important work before 1964 is given by Winter and Christensen (1964). The uptake rates and steady state accumulation of 14C-amino acids under various conditions have been in-vestigated and compared. Winter ( 1962) and Winter and Christensen (1962) studied transport into erythrocytes suspended either in plasma of heparinized blood or in Rak-er's medium. Their results distinguished between initial rates of entry and steady state intracellular amino-acid-accumulation. Contrary to the following order of initial rate of entry L-leucine > . . . . > L-alanine > . . . . > glycine, and after a long time of incubation, glycine * DR. RICHARD MOORE received the Ph.D. degree in Bio-physics from the University of Rochester, N.Y., and a D.Sc. in Bioengineering from George Washington Univer-sity, Washington, D.C. Since 1969, he has been an As-sociate Professor in the Department of Laboratory Medi-cine of the University of Minnesota. ** DR. RITA PARENTI received a Ph.D. in Biochemistry from the University of Milan. Her professional interests include membrane transport. ***I. RENA B1z10s is in the department of Laboratory Medicine at the University of Minnesota. Journal of, Volume Thirty-seven, Nos. 2 and 3, 1971 showed the highest intracellular accumulation, for ex-ample. Furthermore, Winter and Christensen reported a direct relation between transport rates and the size of the apolar hydrocarbon side chain of the entering amino acid. In terms of kinetics they classified three modes of uptake: a mediated and easily saturable type for long chain amino acids; a second nonsaturable type for amino acids with large hydrocarbon side chains; and a third, low-capacity, saturable type for alanine and glycine. Fast counter-transport was observed for the amino acids that use the leucine-type transport system. Comparable re-sults were reported by Oxender (1962) in connection with the amino acid transport into Ehrlich ascites tumor cells. Bjornesjo (1964) studied (what appears to be steady state) accumulation of amino acids in erythrocytes sus-pended either in plasma or in TRIS-HCl buffer. His re-sults showed high uptake for glycine, phenylalanine, and lysine, intermediate for tryptophan, and negligible up-take for glutamic acid. A comparative experimental study was conducted to determine the effect (if any) of freezing and thawing on the amino acid transport into erythrocytes. Materials and methods of collecting The 14C-amino acids used in this study were obtained in a highly purified grade from the New England Nuclear Corporation (Boston, Massachusetts). Human blood was collected from healthy adult donors in polyethylene bags containing 75 ml. of citric acid-sodium citrate-dextrose anticoagulant per 445 ml. of venous blood. The erythrocytes were separated qualita-tively by centrifugation (5,000 x g for 10 minutes). Sev-eral aliquots of packed red cells were stored overnight at 4 C, while several others were mixed with glycerol ( 15 percent by volume as a cryoprotective agent; Pert, et al., 1963; 1965), and were frozen and stored overnight at -170 C. Samples of the erythrocytes stored at 4 C were washed with isotonic TRIS-HCl buffer (pH 7.6) containing 0.1 M NaCl, 0.039M KCl, 0.005M MgC1 2 , 0.001M phos-phate buffer, and 0.044M glucose. After three washings the packed erythrocytes were suspended in isotonic TRIS-HCl buffer solution (final pH 7.4; haematocrit 119 20-25%; haemoglobin l0gm/100 ml.) containing 0.2 mM 14C-amino acid. The 14C-amino acid-erythrocyte suspension was incu-bated at 3 7 C in a Dubnoff Shaking. Incubator {New Brunswick Scientific Corporation) with continuous, gen., tle (90 rpm) shaking to assure uniform mixing. Samples (10 ml.) were withdrawn at various time intervals (3 min., 30 min., 1, 2, 3, and 4 hours) during the incuba-tion. "Separaid" polystyrene beads (Unitech Chemical Manufacturing Company) were added, and the samples were centrifuged at 1,200 x g for 10 minutes; A 500 µ,l. sample of the supernatant medium, and a weighted 0.5 ml. sample of the packed erythrocytes were taken. The 14C-amino-acid-containing erythrocytes were lysed in 1 ml. of distilled water and the proteins were precipi-tated with the addition of 3.5 ml. of 10 percent cold trichloracetic acid. The sample was kept in an ice bath for 30 minutes, was centrifuged, and then 0.5 ml ali-quots of the 14C-amino-acid-containing supernatant were transferred into low-potassium-glass vials each one con-taining 15 ml. of fluor (1,000 ml. of dioxane, 100 gm. of naphthalene, 7 gm. of PPO and SO mg. of POPOP). The 500 µ.,l. samples of the supernatant medium were treated in a similar way. All samples were analyzed for radioactivity with a dual channel beta spectrometer (Packard Tri-Carb Model 3203). Each sample was counted until two suc-cessive counts agreed within 3 % . At least four or five runs. were made fqr each amino acid under investigation. The frozen erythrocytes were thawed in warm water ( 45 C) and were washed once with isotonic normal sa-line-sucrose (30 percent by weight), and once with iso-tonic normal saline-mannitol (5 percent by weight) solu-tions. The procedure described in connection with the samples of erythrocytes stored at 4 C was then repeated: Treatment of Data The data obtained were two complimentary sets of counts of radioactivity (intracellular and extracellular) in terms of concentration of 14C-amino acid per gram of sample. These values were corrected (by subtraction) for the counting rate of the background. Further correc-tions were made for: the plasma trapped between the packed cells; the non-solvent volume of the cells; cell density; and cell volume changes taking place during the experiments. The final values were averaged for the four or five experimental runs for each amino acid. In analyzing the data, the following graphs were plotted: (i) radioassay values versus time of incubation; (ii) logarithm of the difference between the intracellular concentration of each amino-acid at a given time during incubation and at infinite time (log x) versus time; (iii) AX versus time; and (iv) reciprocal Ax versus time. A Lineweaver-Burk plot also was attempted. The relative rates of 14C-amino-acid uptake foto ery-throcytes were found to be (in decreasing order) : phenyl-alanine, leucine, alanine, trytophan, glycin .. The freeze-thaw process used had no significant effect on either the above order or on the magnitude of the rate constants of the rate constants of the amino acid uptake (Table I) . 120 The graphs of radioassay values versus time of incu-bation gave curves which appeared to be monotonic func-tions with a single exponential term. . · i · The half-times required to reach the equilibrium con-centration. were determined graphically. The half-time . va.lµes obtained fro!Jl intracellular and extracellular radio-activity data were very close together. The rate of in-tracellular radioactivity increase, though, was more ac-curate because in that case the radioactivity varied from an initial zero concentration to some finite value ( at the end of the incubation interval), instead of being a differ-ence between two large numbers. The graphical analysis of the data showed that the transport reaction could not be described by either zero or second order kinetics. Furthermore, it could not be described by a Michaelis-Menten equation. The reaction or, at least, the rate limiting reaction, appeared to be of first order. The calculated values of that rate constant (k) are given in Table I. TABLE I - Correspondence between rates of entrance and amino acids into control or into frozen-thawed human erythrocytes. Half-time to reach steady state, minutes Amino Acid Control• Frozen-thawed PHENYLALANINE . . . . . . . . . . . . . . . 7 7 LEUCINE . . . . . . . . . . . . . . . . . . . . . . . . 26 12 ALANINE . . . . . . . . . . . . . . . . . . . . . . . . 35 35 TRYPTOPHAN . . . . . . .. . . . . . . . . . . . . 50 50 GLYCINE ........................ 145 120 *The control cells were non-frozen cells. The results of amino acid uptake into erythrocytes and into frozen-thawed erythrocytes show that the freezing and thawing process used in the experiments did not dis-turb this aspect of cell physiology. These observations are in agreement with the work of Wallach, et al. (1962), who used a similar freezing-thawing method and found no change in the cation flux and electrolyte concentration of erythrocytes. · The order of the relative rates of amino:..acid uptake is in good agreement with the order of entry rates reported by Winter and Christensen ( 1964). Table II shows that the observed rate of transport de-creased as the molecular weight of the respective amino acid increased. This observation supports the conclusions of Ox.ender, Winter, and Winter and Christensen in con-nection with the steric relationships involved in amino acid transport. Diffusion did not appear to be the primary TABLE ·u - Comparison oi the variation of the rate of entrance with the variation of the molecular weight of the amino acids. Half-time for Molecular Amino Acid control cells, minutes Weight PHENYLALANINE . . . . . . . . . . . . . . . 7 LEUCINE ........................ 26 ALANINE ........................ 35 TRYPTOPHAN . . . . . . . . . . . . . . . . . . . 50 GLYCINE ........................ 145 165 131 89 204 75 ·. The·MinnesotaAcademy of Science mode of transport in the present study; amino acid trans-port might thus be dependent on carriers in the cell mem-brane as proposed by Oxender, _Chri~tensen, and :their associates. Acknowledgments This work was supported in part by Public Health Service Grant RR-00267 from the Division of Research Resources, by Grant SMF-18-70 from the Minnesota Medical Foundation and by a grant from the Graduate School of the University of Minnesota. Some of· this work was done while authors Moore .and Parenti were at the blood program research laboratory of the Ameri-can National Red Cross in Washington, D.C. The authors acknowledge with gratitude the contribu-tions of the following organizations and individuals: The American Red Cross Chapter, Washington, D.C., for providing the human erythrocytes; Mr. Paul K. Schork for helping with the freezing-thawing process; Dr; James H. Pert, for helpful discussions; and Miss Colleen Boschert and Miss Mary Scherman, for typing the manuscript. References BJoRNESJo, K. B.: Uptake of labeled amino acids into human erythrocytes in vitro. Clin. Chim. Acta 11, 197-204, 1965. Journal of, Volume Thirty-seven, Nos. 2 and 3, 1971 CHRISTENSEN, H. N.: Methods for distinguishing amino acid transport systems of a given cell or tissue. Fed. Proc., 25:850-853, 1966. OXENDER,-D. L.: Nature of amino acid "exit from cells. Fed. Proc. 21:148, 1962. PERT, J. H., ScHORK, P. K., and MOORE, R.: A new · · method of low temperature blood preservation using liquid Nitrogen and a Glycerol-Sucrose additive. Clin. Res., 11: 197, 1963. PERT, J. H., MooRE, R. ScHORK, P. K.: Low temperature preservation of human erythrocytes. Bib. Haem. 23:-674-682, 1965 .. WALLACH, S., ZEMP, J. W., CAVINS, J. A., JENKINS, L. J., JR., BETHEA, M., FRESHETTE, L., HAYNES, L. L., TULLIS, J. L.: Cation flux and electrolyte composition of frozen - deglycerolized blood. Blood 20:344-353, 1962. WINTER, C. G.: Relative transport affinity of amino acids . in human erythrocytes. Fed. Proc., 21:148, 1962. WINTER, C. G. and CHRISTENSEN, H. N.: Migration of amino acids across the membrane of the human erythrocyte. J. Biol. Chem., 239:872-878, 1964. 121 

Effect of Freezing and Thawing on Uptake of Amino Acids into Human Erythrocytes

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Effect of Freezing and Thawing on Uptake of Amino Acids into Human Erythrocytes

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