J Clin Pathol: first published as 10.1136/jcp.40.10.1182 on 1 October 1987. Downloaded from J Clin Pathol 1987;40:1182-1186 Oxpentifylline and cetiedil citrate improve deformability of dehydrated sickle cells J STUART, P C W STONE, YY BILTO, A J KEIDAN From the Department ofHaematology, Medical School, University ofBirmingham, Birmingham SUMMARY Erythrocytes from 14 patients with homozygous sickle cell anaemia were treated with the calcium ionophore A23187 to induce loss of cellular potassium and water. The dehydrated cells showed a decrease in filterability (loss of deformability) through pores of 5 gm diameter. Ox- pentifylline and cetiedil citrate, which preserve erythrocyte cation and water content, had a significant (p < 0-01) protective effect against loss of deformability at a concentration of 1 gmol/l. Oxpentifylline showed no adverse effect on the rheology, morphology, or haemolysis of sickle cells at concentrations up to 500 jmol/l. Drugs that act on the erythrocyte membrane to maintain cell hydration are of potential rheological benefit in sickle cell anaemia. The dense erythrocytes in patients with sickle cell ana- 14 patients with homozygous (SS) sickle cell anaemia. emia are depleted of potassium' and the associated Oxpentifylline was compared, at equimolar concen- loss of cell water contributes to their high mean cell trations, with the iminoester cetiedil citrate which alsocopyright. haemoglobin concentration (MCHC).2 4 Increase in changes the cation content ofsickle cells by increasing MCHC greatly increases the intracellular poly- passive influx of sodium or preventing loss of potas- merisation of haemoglobin S (HbS)s so that polymer sium induced by calcium gain. 17 - 20 Intravenous may form at arterial oxygen tension.6 infusion of cetiedil has been shown to shorten the It is not known whether sickle cells lose potassium duration of vaso-occlusive crisis.2' and water by direct leakage through a cell membrane distorted by extended spicules of polymer,7 or by Material and methods opening the Gardos8 channel in response to transient http://jcp.bmj.com/ increases in cytoplasmic calcium before this cation be- The 14 SS patients were in the asymptomatic steady comes compartmentalised in membrane vesicles.9 Al- state. Heparinised venous blood was washed through ternatively, young sickle cells that swell in response to Imugard IG 500 cotton wool (Terumo Corporation, a fall in pH at sites of ischaemia may lose potassium Tokyo, Japan), using 20 mmol/l HEPES buffered sa- and water via the recently described volume sensitive line (HBS) of osmolality 290 mmol/Kg H20 and pH KCI cotransport system.'0" Whatever the cause of 7 4, to give a suspension of leucocyte free washed their dehydration, the deformability of sickle cells is erythrocytes.22 Passage through cotton wool does not on September 27, 2021 by guest. Protected adversely affected by the increase in MCHC122" and selectively remove irreversibly sickled cells23 or other is substantially improved by rehydration.12 14 dense sickle cells,24 and HBS has been shown not to Oxpentifylline is a dimethyl xanthine derivative change the MCHC of sickle cells.25 that binds reversibly to erythrocyte membranes Filtration of oxygenated sickle cells through poly- and has been shown to increase walking distance in carbonate membranes with pores of 5 g.m diameter patients with atherosclerotic intermittent claudica- and 10-I Ilm length (Nuclepore Corporation, Pleas- tion.'5 We have recently shown that the drug pre- anton, California, USA) was measured at 37°C using vents loss of potassium and therefore water from a Hemorheometre (IMH, 95470 St Witz, France) to normal (haemoglobin AA) erythrocytes that had been give an index of filtration (IF) corrected for erythro- loaded with calcium using the ionophore A23187.16 cyte count.'6 This index is the ratio of the flow re- The same experimental model has now been used to sistance of erythrocytes suspended in HBS at a cell study the action of oxpentifylline on sickle cells from count of 0 3 x 1012/I to that of HBS alone, and is expressed as relative resistance rather than absolute Accepted for publication 21 December 1987 units; an increase in IF indicates loss of erythrocyte 1182 J Clin Pathol: first published as 10.1136/jcp.40.10.1182 on 1 October 1987. Downloaded from Improving the deformability ofdehydrated sickle cells 1183 (841) 100 80 560- 60- 480- & 40- c ;2020- 6400° so I .3- 0 320 0< w K 0- 1-- .3 ST 0 240- ;-40 -a a" * - : -60- -3 160 01~ . - -80 -100 1 10 100 500 1 lb 160 5so 0- Oxpentifylline (prnol/l) Cetiedil (pmol/I) Buffer A 23187 Fig 2 Index offiltration ofsickle cells after incubation for Fig I Index ojfiltration (IF) ofsickle cells from 14 60 minutes in buffer containing A23187 ionophore and either patients after incubation ofcells for 60 minutes in control oxpentifylline or cetiedil citrate. Results (mean and 95% buffer and in buffer with calcium ionophore A23187. confidence intervalfor 14 patients) expressed as percentage copyright. Increase in IF indicates loss offilterability. change from IF valuesfor sickle cells incubated wilh A23187 (fig 1) but without either drug. deformability. A pore diameter of 5 gm rather than 3 sessed by interference microscopy on 300 oxygenated gm was used as this enhances sensitivity to cyto- cells fixed with 125% w/v glutaraldehyde in HBS, plasmic viscosity and therefore to cytoplasmic hy- using the Bessis classification29 for stomatocytes and dration.2627 The polycarbonate membranes were echinocytes. Cells whose length was twice their width cleaned by ultrasonication in 1% w/v sodium dodecyl or those which had angular contours were counted as http://jcp.bmj.com/ sulphate and reused.28 irreversibly sickled cells (ISC).30 The extent of hae- A stock solution of the calcium ionophore A23 187 molysis of the erythrocyte suspensions was assessed (Calbiochcm brand, Behring Diagnostics, La Jolla, from the supernatant haemoglobin concentration, California, USA) in absolute ethanol ([-9 mmol/l) measured spectrophotometrically using tetramethyl was stored at -40°C and diluted in ice cold HBS benzidine. Significance (two tail) was determined by immediately before use. Oxygenated sickle cells Wilcoxon's non-parametric signed rank test for (0-3 x 1012/I) were incubated for 60 minutes at 37°C paired data. on September 27, 2021 by guest. Protected in control buffer (HBS, CaCI2 100 jmol/l, MgCl2 2 mmol/l, glucose 10 mmol/l) and in control buffer plus Results A23187. As there was considerable individual vari- ation in response to ionophore a concentration of Calcium loading of sickle cells using A23187 iono- A23 187 (range 0 5--13 pmol/l) which increased the IF phore caused an increase in IF (loss of filterability) value by at lcast two-fold, was selected for each pa- after 60 minutes compared with cells in control buffer tient. Oxpentifylline or cetiedil citrate, at final drug (fig 1). As there was considerable variation between concentrations of 0, 1, 10, 100 and 500 gmol, was patients in the extent to which A23187 increased IF, added to the cell suspensions at 37°C, 15 minutes be- the effects of oxpentifylline and cetiedil citrate were fore the addition of ionophore. Oxpentifylline was expressed as pcrcentage change from the IF value af- dissolved in HBS immediately before use. A stock ter incubation with A23 187 alone (fig 2). All four con- solution of cetiedil citrate in absolute ethanol (125 centrations of oxpentifylline significantly improved mmol/l) was stored at -40°C. IF (10 pmol/l, p < 0-05; other concentrations, Morphology of incubated erythrocytes was as- p <0 01) and caused no change in erythrocyte mor- J Clin Pathol: first published as 10.1136/jcp.40.10.1182 on 1 October 1987. Downloaded from 1184 Stuart, Stone, Bilto, Keidan Table Effect ofoxpentifylline and cetiedil on erythrocyte of small amounts of intracellular polymer in sickle morphology and haemolysis after incubation with A23187 erythrocytes at arterial oxygen tension.34 ionophore for 60 minutes (mean and SEM, n = 14) Cetiedil is a vasodilator" that enhances passive en- Irreversibly Supernatant try of sodium into erythrocytes17 18 and decreases po- Stomatocytes Echinocytes sickled cells haemoglobin tassium loss via the Gardos pathway after calcium (%) (%) (%) (mg/I) loading.19 20 Both mechanisms could increase cell Control buffer 49 8-1 9.7 97 water, reduce MCHC and cytoplasmic viscosity, and (1 9) (1.4) (2-1) (7) thus improve erythrocyte deformability. Intravenous A23187 35 19l1t 83 101 cetiedil (0-4 mg/kg body weight) has been shown to (1-2) (4-9) (2-2) (4) Oxpentifylline: shorten the duration of vaso-occlusive crisis,21 and a 1 ,mol/1 3-7 17-4 9-3 107 single infusion at this dose gives a peak plasma con- (1-0) (3-0) (2 5) (8) 10 jmol/I 4-2 16-3 8-6 114 centration of 025 jsmol/l.36 A previous in vitro (1 1) (4-1) (2-0) (7) filtration study of deoxygenated sickle cells showed a 100 1mol/I 5-3 11-4 9.3 99 (1.7) (2-9) (2-0) (3) beneficial effect of cetiedil at 50-200 jmol/l, with no 500 jmol/I 4 5 13.0 8-4 94 effect on oxygenated cells.37 The higher sensitivity of (2-9) (2 8) (2-0) (5) our filtration technique detected an effect on oxygen- Cetiedil: I Sumol/1 59 13-1 8-9 97 ated SS cells treated with ionophore at I pmol/l, the (2-1) (2-9) (2 0) (5) lowest concentration studied. At 100 jimol/l and 10lmol/l 8-4* 8-1* 8-2 110 (2-0) (1-8) (1-8) (6) above, the drug caused substantial cell swelling (sto- 100 imol/I 70-1* 2.1* 5-0 1537* matoxytosis) and haemolysis, as previously re- (4 9) (0 8) (0 9) (684) 500 mol/l 99-1* 0* 0-8* 11316* ported.17 18 (0-3) (0-3) (104) Oxpentifylline has recently been shown to reduce loss of potassium and therefore water from normal tp < 0 01 compared with buffer value.