Effects of Glyceraldehyde on the Structural and Functional Properties of Sickle Erythrocytes Alan M. Nigen, James M. Manning J Clin Invest. 1978;61(1):11-19. https://doi.org/10.1172/JCI108909. Research Article The d- and l-isomers of glyceraldehyde are equally effective in the inhibition of SS erythrocyte sickling in vitro. The following compounds at a concentration of 20 mM were ineffective in inhibiting sickling: glyceraldehyde-3-phosphate, d- erythrose, d-ribose, d-fructose, d-glucose, d-sucrose, dihydroxyacetone, and methylglyoxal. Glyceraldehyde does not reverse the sickling of cells in the deoxy state. The properties of purified hemoglobin after treatment with glyceraldehyde and of the hemoglobin isolated from treated cells are very similar; these results suggest that glyceraldehyde itself is the reactive species within the erythrocyte. Erythrocyte glutathione is decreased by treatment in vitro with the aldehyde. Relatively high concentrations of glyceraldehyde (50 mM) lead to a small amount (3%) of cross-linking between hemoglobin monomers as well as to some cross-linking of erythrocyte membrane proteins. Lower concentrations of dl- glyceraldehyde (5-20 mM), which reduce the sickling of erythrocytes in vitro, lead to proportionally less cross-linking of hemoglobin. Cells that have been treated with those concentrations of the aldehyde show little change in their osmotic fragility, exhibit improved filtration properties, and have a lowered viscosity. Find the latest version: https://jci.me/108909/pdf Effects of Glyceraldehyde on the Structural and Functional Properties of Sickle Erythrocytes ALAN M. NIGEN and JAMES M. MANNING, The Rockefeller University, New York 10021 A B S T R A C T The D- and L-isomers of glyceralde- the treated erythrocytes. In the present communica- hyde are equally effective in the inhibition of SS tion we present further studies on the effects of erythrocyte sickling in vitro. The following com- glyceraldehyde on hemoglobin and on some structural pounds at a concentration of 20 mM were ineffec- and functional properties of the erythrocyte. tive in inhibiting sickling: glyceraldehyde-3-phos- phate, D-erythrose, D-ribose, D-fructose, D-glucose, METHODS D-sucrose, dihydroxyacetone, and methylglyoxal. Glyc- Whole blood from patients homozygous for sickle cell eraldehyde does not reverse the sickling of cells anemia was collected into heparinized tubes by venipunc- in the deoxy state. The properties of purified hemo- ture and was used within 48 h; informed consent was ob- globin after treatment with glyceraldehyde and of tained in all cases. Crystalline DL-glyceraldehyde, sodium the hemoglobin isolated from treated cells are very borohydride, dihydroxyacetone, methylglyoxal, glyceralde- hyde-3-phosphate, D- and L-glyceraldehyde, and D- similar; these results suggest that glyceraldehyde fructose were obtained from Sigma Chemical Co., St. Louis, itself is the reactive species within the erythrocyte. Mo. D-Glucose was from J. T. Baker Chemical Co., Erythrocyte glutathione is decreased by treatment Phillipsburg, N. J., D-ribose from Fluka, Basel, Switzerland, in vitro with the aldehyde. sucrose from Mallinckrodt Inc., St. Louis, Mo., and -erythrose Relatively high concentrations of glyceraldehyde from P-L Biochemicals, Inc., Milwaukee, Wis. D_['4C]Glyc- eraldehyde (10.5 mCi/mmol) was from New England Nuclear, (50 mM) lead to a small amount (3%) of cross- Boston, Mass. All other chemicals were reagent grade. linking between hemoglobin monomers as well as In vitro sickling experiments. These experiments were to some cross-linking of erythrocyte membrane pro- carried out as described previously (1). teins. Lower concentrations of DL-glyceraldehyde Physical measurements. Oxygen dissociation curves of whole cells and of isolated hemoglobin, and the minimum (5-20 mM), which reduce the sickling of erythro- gelling concentration of isolated hemoglobin, were deter- cytes in vitro, lead to proportionally less cross-link- mined as described previously (1, 2). Viscosity measure- ing of hemoglobin. Cells that have been treated with ments of whole cells were carried out at 37°C with a those concentrations ofthe aldehyde show little change Harkness viscometer fitted with a 0.5-mm bore capillary in their osmotic fragility, exhibit improved filtration (Coulter Electronics, Inc., Hialeah, Fla.); the shear rate for the experiments was >100/s (3). Before the measurement properties, and have a lowered viscosity. of viscosity, erythrocytes were resuspended in phosphate- buffered saline (PBS),1 pH 7.4 (4), to a hematocrit of 40%. INTRODUCTION For measurement of the viscosity of deoxygenated samples, cells (2.0 ml) were equilibrated with water-saturated nitrogen Recently, we reported that DL-glyceraldehyde in- in a chamber at 37°C for 30 min; a few grains of sodium hibited the sickling of S/S erythrocytes in vitro (1). dithionite were then added to ensure complete deoxygena- tion. After an additional 5 min of equilibration with nitrogen, This aldehyde reacts with hemoglobin S to produce the sample was transferred anaerobically to the viscometer. a significant increase in the minimum gelling con- For measurement of the viscosity of oxygenated cells, centration of the deoxygenerated protein with minimal equilibration was carried out at 37°C for 10 min before trans- effects on the oxygen equilibrium parameters. In those fer to the viscometer. All values are reported as relative studies we surveyed only morphological changes in viscosity compared to water whose viscosity had been meas- ured under the same conditions. Between four and five meas- urements were made with each 2.0-ml sample and the values Dr. Nigen's current address is University of Miami School were averaged. of Medicine. Reprint requests can be addressed to Dr. In experiments where the deformability of erythrocytes Manning at The Rockefeller University. Received for publication 21 July 1977 and in revised form IAbbreviations used in this paper: Hb, hemoglobin; PBS, 12 September 1977. phosphate-buffered saline; SDS, sodium dodecyl sulfate. The Journal of Clinical Investigation Volume 61 January 1978 11-19 11 was measured by filtration through micropore filters, of the isomers of glyceraldehyde shown in Table I washed erythrocytes, either untreated or treated with enantiomers glyceraldehyde, were resuspended at a concentration of clearly indicates that the of glyceralde- 0.5% in PBS that contained 400 mg of bovine serum hyde are equally effective antisickling agents in vitro. albumin/100 ml. For each determination, samples were first Dihydroxyacetone, the tautomer of glyceraldehyde, equilibrated with air or with a venous gas mixture (90.4% does not affect erythrocyte sickling. Glyceraldehyde- N2, 3.8% 02, 5.8% CO2) for 30 min at 37°C with stirring. 3-phosphate, a product of glyceraldehyde metabolism Deoxygenated samples were transferred by positive pressure from the equilibration vessel through Saran tubing into a within the erythrocyte (10), exhibits no effect upon filtration vessel (15-ml glass vessel; Millipore Corp., Bed- sickling, perhaps because it does not cross the erythro- ford, Mass.) that was jacketed at 37°C and also flushed cyte membrane. with a venous gas mixture. On top of the fritted glass filter Studies on a number of other related compounds holder was a polycarbonate filter (Nuclepore Corp., Pleasan- showed that sugars of chain length longer than ton, Calif.) (25 mm in diameter with 5-,um pores). A graduated cylinder was adapted for collection of the filtrate and for glyceraldehyde such as D-erythrose, D-ribose, D-fruc- application of a partial vacuum of 9 cm of Hg by a water tose, and D-glucose, all of which occur predominantly aspirator with an on-line regulator valve to attain the de- in the hemiacetal form, have no effect upon sickling sired negative pressure. Filtration was initiated by opening at a concentration of 20 mM. Sucrose, a nonreducing a stopcock attached to a vacuum line and the time for filtra- tion of all of the suspending medium was measured with a disaccharide, is also ineffective at this concentration. stopwatch. The filtered cells were then collected and centri- Methylglyoxal has been shown to be formed slowly fuged; the amount of lysis was determined by measure- from glyceraldehyde in solution (11), but treatment of ment at 540 nm of any hemoglobin in the supernatant erythrocytes with this compound resulted in cell lysis solution and of the amount of hemoglobin in the packed and oxidation of hemoglobin. Acetaldehyde had no erythrocytes. Determinations of osmotic fragility were performed as effect upon erythrocyte sickling (data not shown). described by Emerson et al. (5). Samples were adjusted To test whether glyceraldehyde could restore nor- to a hematocrit of 40% before addition of the sodium mal morphology to sickled cells in the deoxy state, chloride solution. erythrocytes were equilibrated for 30 min at 37°C Gel electrophoresis. Sodium dodecyl sulfate (SDS) poly- acrylamide gel electrophoresis of isolated proteins was with the venous gas mixture before anaerobic addition carried out as described by Weber and Osbom (6) with 7.5 of glyceraldehyde and incubation for an additional 90 or 10% cross-linked gels. The electrophoretic system used min. After fixation of the cells with formalin, the to study erythrocyte membrane proteins was similar to that results shown in Table II were obtained. Clearly, described by Fairbanks et al. (7), except that 5% cross- linked gels were used. After staining with Coomassie Blue, the amount of protein in each band was measured at 600 TABLE I nm on a Gilford 222 spectrophotometer (Gilford Instrument Effect of Sugars and Related Compounds on the Laboratories Inc., Oberlin, Ohio) equipped with a model 2520 Sickling of Erythrocytes gel scanner. Hemoglobin purification. Hemoglobin S from patients Cells with homozygous for sickle cell anemia was purified on a column Compound normal morphology (2 x 30 cm) of DE-52 cellulose equilibrated with 0.05 M Tris buffer, pH 8.5; a linear gradient of 0.05 M Tris, pH 8.3 (500 ml) and 0.05 M Tris, pH 7.3 (500 ml) was used to elute the protein (8).