An inhibitor of the sodium pump obtained from human placenta

T H E LAN C ET Early report An inhibitor of the sodium pump obtained from human placenta P J Hilto n, R W White , G A Lo rd, G V Garne r, D B Go rdo n, M J Hilto n, L G Fo rni, W Mc Kinno n, F M D Is mail, M Ke e nan, K Jo ne s , W E Mo rde n Summary Background Muc h e ffo rt has be e n e xpe nde d in the s e arc h fo r an e ndo g e no us s o dium/ po tas s ium inhibito r pump, a of the c o mpo und c e llular of majo r phys io lo g ic al impo rtanc e , whic h has be e n implic ate d in the me c hanis m of e s s e ntial hype rte ns io n. Othe rs have s ug g e s te d that o uabain o r an is o me r o f o uabain may be the e ndo g e no us pump inhibito r. Ne o natal c o rd s e rum c o ntains an inhibito r o f the s o dium pump; we atte mpte d to is o late and c harac te ris e this s ubs tanc e fro m human plac e ntas . M ethods Ho mo g e nis e d plac e ntas we re dialys e d and the re s ulting s o lute s we re trappe d o n o c tade c yls ilyl s ilic a and the n s e parate d by hig h-pe rfo rmanc e liquid c hro mato g raphy. Me as ure me nt o f the ac tivity o f the s o dium pump o f human le uc o c yte s was us e d to te s t e ac h frac tio n fo r the pre s e nc e o f the inhibito r. Findings An inhibito r o f the s o dium pump was o btaine d by this te c hnique in a mas s s pe c tro me tric ally pure fo rm with a mas s o f 3 7 0 Da, an e mpiric al fo rmula o f C 2 4 H3 4 O3 and o nly o ne hydro xyl g ro up. The c harac te ris tic frag me ntatio n patte rn o bs e rve d in ne g ative -io n mas s s pe c tro me try was c o mpare d with tho s e o f vario us mo de l c o mpo unds ; this c o mparis o n s ug g e s te d that the ac tive mate rial was a dihydro pyro ne -s ubs titute d s te ro id. Interpretation The s e re s ults s ug g e s t that a dihydro pyro ne s ubs titute d s te ro id is an e ndo g e no us re g ulato r o f the s o dium pump in humans and, pre s umably, o the r mammals . Pro o f of the e ndo g e no us o rig in will re quire the de mo ns tratio n o f a pre vio us ly unre c o g nis e d bio s ynthe tic pathway. Lanc e t 1 9 9 6 ; 3 4 8 : 3 0 3 –0 5 Introduction T here is indirect evidence for hormonal regulation of the mammalian cellular sodium/potassium pump. T he natriuresis of volume expansion during which plasma develops the ability to inhibit the sodium pump, the reduced activity of the sodium pump in patients with uraemia and essential hypertension, and the transferability of this property by plasma from such patients support the existence of such regulators. 1–8 de Wardener and M acG regor 9 have argued that in essential hypertension the presence of an inhibitor of the sodium pump in plasma is central to the pathophysiology of the condition. M any attempts have been made to characterise the endogenous substance or substances that inhibit the sodium pump, 10–14 mostly with inconclusive results. T he report of M athews and colleagues14 that the mass spectral characteristics of ouabain can be detected in a concentrate of human plasma aroused great interest and controversy. If ouabain is an endogenous inhibitor of the sodium pump it must be synthesised by mammals. Such biosynthesis would require several previously unrecognised pathways in man. H owever, there have been suggestions that what is measured in human plasma by a radioimmunoassay based on ouabain antibodies is not authentic ouabain. T his view is based on the radically different retention times of ouabain and the immunoreactive peak on reversed-phase highperformance liquid chromatography (H PLC ). 15,16 Laredo et al17 claim, however, that with their antibody to ouabain, the immunoreactivity of plasma is completely congruent with the retention time of ouabain. D espite the failure to resolve this issue, reports continue to appear describing changes in the concentration of “ouabain” in human plasma under various clinical and experimental conditions. T he conclusions are based solely on immunoassay and ignore the possibility that what is being measured is a substance cross-reacting with the antibody to ouabain. A commercially available ouabain assay kit (N ew England N uclear, Wilmington, D elaware, U SA) quotes a cross-reactivity to acetylstrophanthidin of 171% and to digitoxin of 48%; thus, there is a need for caution in reporting concentrations of ouabain in human plasma measured by this technique. Antibodies raised to the toad-derived inhibitors of the sodium pump (the bufadienolides), with low cross-reactivity to ouabain, also detect a rise in concentrations of an immunologically reactive substance during volume expansion. 18 T he starting point for these experiments was the demonstration that unmodified human plasma from uraemic, hypertensive, and volume-expanded patients inhibits the sodium pump of circulating cells. T he concentrations of ouabain reported in human plasma measured under any conditions by immunoassay fall short by more than an order of magnitude of those necessary to account for the previous experimental results. We have previously reported the presence of a factor in human umbilical cord serum that inhibits the sodium pump of leucocytes. 19 We now present details of the recovery and identification of the substance responsible for this activity in human placentas. St Thomas’ Hospital, London SE1 7 EH ( P J Hilto n MD, W Mc Kinno n BSc ) ; M anchester M etropolitan University and M ichael Barber Centre for M ass Spectrometry, University of M anchester Institute of Science and Technology ( G A Lo rd PhD, G V Garne r PhD, Pro f D B Go rdo n PhD, W E Mo rde n CChe m ) ; Department of Chemical Sciences, University of Hertfordshire, Hatfield ( F M D Is mail PhD) ; and Department of Chemistry, King’s College, London, UK ( M Ke e nan BSc , K Jo ne s PhD) R W White BSc , M J Hilto n PhD, L G Fo rni Correspondence to: Dr P J Hilto n Vol 348 • August 3, 1996 PhD, Methods Placentas were obtained immediately after delivery and treated as described below or frozen at ⫺20°C . F our placentas per batch were homogenised in 10% (by volume) methanol in water and sodium azide (0·1% weight/volume). T he homogenate was dialysed in Visking tubing (pore size 2·4 nm) for 48 h at room temperature against 10% methanol in distilled water containing 50 g octadecylsilyl (OD S) silica. After washing with water the 303 O 2 9 ·0 2 3 ·0 OH OH HO OH 3 9 7 ·3 3 9 3 ·3 60 [ M+Na] + 3 , 1 4 , 2 0 :2 1 – bufenolide O CH3 [ M+H] + CH3 3 6 5 ·3 [ M+K] + 3 6 9 ·3 3 7 1 ·3 O 80 20 CH3 CH3 O 1000 40 CH3 CH2 L-rhamno s e 100 OH HO 0 B O O OH 3 9 ·0 1 3 1 ·0 20 O Bufalin 4 0 9 ·3 % re lative inte ns ity 40 2 7 7 ·2 4 0 9 ·3 60 5 7 ·0 80 Ouabain 4 5 ·0 100 9 3 ·1 A 7 5 ·1 1 8 5 ·1 T H E LAN C ET 0 HO 430 425 420 415 410 405 400 395 390 385 380 375 370 365 m/ z Fig ure 1 : Positive-ion FAB mass spectrum of biologically active Fig ure 2 : Structures of ouabain (a cardenolide), bufalin (a bufadienolide), and the synthetic bufenolide 3 ␤, 1 4 ␣, 2 0 :2 1 -bufenolide material Sig nific ant io ns s e e n at m/ z 4 0 9 , 3 9 3 , and 3 7 1 . Othe r pe aks are de rive d fro m g lyc e ro l FAB matrix. Spe c trum B is an e xpans io n o f the re g io n 3 6 5 –4 3 0 o f s pe c trum A. OD S silica was eluted with 100% methanol. T he eluate, after evaporation to dryness, was taken up in 100% methanol and subjected to reversed-phase H PLC on an OD S silica column (0·75 cm internal diameter, 25 cm length) with a continuous gradient over 2 h (10% methanol/90% water to 30% methanol/70% water, flow rate 2·5 mL/min). F ractions were collected every 2 min, evaporated to dryness, and analysed. T he biological activity of each fraction was assessed by its ability to inhibit the sodium pump of human leucocytes. 20 Briefly, leucocytes were separated from whole blood by differential sedimentation followed by hypotonic lysis of residual erythrocytes. T he leucocytes were loaded with sodium-22 while simultaneously being exposed to the fraction under test; extracellular 22N a was removed by washing, and the loss of the labelled sodium from the cells was observed over 12 min in the presence and absence of ouabain. T he ouabain-sensitive rateconstant for the loss of 22N a represented the activity of the sodium pump. Results Inhibition of the sodium pump of leucocytes was observed in the fraction that eluted at 82 min in a nominal methanol concentration of 24%. On positive-ion fast atom bombardment (F AB) mass spectrometry, the principal ion of the active material was of m/z (mass/charge ratio) 409. Peaks were also identified with m/z of 393 and 371 (figure 1). T hese correspond to the potassiated, sodiated, and protonated ions of a substance of integer mass 370 D a. T he accurate mass of the potassiated ion was 409·215 D a; from this value can be derived an empirical formula of C 24H 34O 3. T his empirical formula, with its low H /C ratio is in keeping with a cyclic compound, almost certainly a steroid. D euteration of the molecule increased the mass by 1, which indicates the presence of a single hydroxyl group. N egative-ion mass spectrometry showed a prominent deprotonated molecule ion of m/z 369, in keeping with the integer mass of 370. T his yielded product ions of m/z 97 and 80, the latter being consistent with loss of a hydroxyl radical. Interestingly, the mass spectrum reported by M athews et al14 to show the presence of ouabain also has features that are consistent with our findings of material of mass 370 (prominent peaks at m/z 371, 393, and 409). 304 T o investigate further the characteristic fragment of m/z 97, we examined model compounds, derived principally from cardenolides and bufadienolides. T he cardenolides are widespread among plants and include digoxin and ouabain, the best-known inhibitors of the sodium pump. Amphibia and some plants synthesise a group of compounds (the bufadienolides) that are also potent inhibitors of the sodium pump and which are structurally similar to the cardenolides. Both are steroids with an unsaturated lactone ring at C 17 (figure 2). Whereas the cardenolides have a five-membered furone ring, the bufadienolides have a six-membered ␣-pyrone ring at C 17. N either the cardenolides ouabain, digoxin, and digitoxin nor the bufadienolide bufalin yielded the fragment of m/z 97. Further chemical manipulation of bufalin was therefore undertaken. Bufalin was first reduced to dihydrobufalin by catalytic hydrogenation. Although activity against the sodium pump of leucocytes was retained, no fragmentation to m/z 97 was seen. We next eliminated the 14-OH group of these compounds by dehydration with phosphorus oxychloride. Although mass spectrometry of this compound showed fragments of m/z 97 and 80, as seen in the active material, biological activity was destroyed. T he last possibility was that a hydrogen atom rather than an OH group or a double bond was present at C 14. T o examine the fragmentation pattern induced by this configuration, a synthetic bufenolide (3␤-OH , 14␣20:21 bufenolide, figure 2) was prepared from androsterone. T his compound not only inhibited the sodium pump of leucocytes but also gave the fragments of m/z 97 and 80 characteristic of the active material in negative-ion mass spectrometry. Discussion Since de Wardener and colleagues1 suggested that a humoral factor other than aldosterone was involved in the natriuresis of volume expansion, this subject has been controversial. de Bold discovered atrial natriuretic peptide (AN P) in 1982, 21 but under conditions of salt overload the natriuretic activity of the plasma was associated with an inhibition of the sodium pump that is not a feature of AN P nor the more recently identified brain and C N P natriuretic peptides. T he demonstration that inhibition of Vol 348 • August 3, 1996 T H E LAN C ET the sodium pump is also a feature of uraemia 7,8 and essential hypertension 4 led to a hypothesis linking salt intake, chronic secretion of the natriuretic hormone, vasoconstriction, and hypertension. 9 Although several groups of investigators reported possible candidate compounds10–13 for the role of endogenous sodium-pump inhibitor, none has found widespread acceptance. H owever, in 1991, M athews and colleagues reported 14 that an extract of 85 L of human plasma contained a compound with the unequivocal mass spectral characteristics of ouabain, and a subsequent report described the isolation of a stereoisomer of ouabain from bovine hypothalamus. 22 T here are difficulties in accepting these plant-derived poisons as legitimate candidates for the role of sodium-pump regulators in animals. One possibility is that they originated in the environment and entered the animal via the food chain, with the sodium pump acting as a concentrating mechanism. Our experiments show that the fragmentation pattern of the active material can be reproduced in semi-synthetic dihydropyrone-substituted steroids, and that this fragmentation does not occur when a hydroxyl group is present at C 14, from which it may be predicted that the active material must have either a hydrogen atom or a double bond at this site. T he presence of a double bond at C 14 results in loss of the power to inhibit the sodium pump. T he conclusion was, therefore, that a hydrogen atom must be present at the C 14 of the placental material. T his idea was supported by study of the compound 3␤OH , 14␣ 20:21 bufenolide, which not only gave the characteristic product ions of the active material but also inhibited the sodium pump of human leucocytes. We conclude that the inhibitor of the sodium pump isolated from human placentas is a dihydropyrone-substituted steroid. Its mass is 370 D a and its empirical formula C 24H 34O 3. T he pyrone ring contains one double bond, rather than two as in the toad poisons (the bufadienolides), and the compound is therefore best described as a bufenolide. T he position of the double bond within the pyrone ring remains unknown. G iven that the material is a bufenolide, the empirical formula mandates an additional double bond within the steroid nucleus. We cannot state categorically that this compound originated in the pregnant women rather than the environment, though we are unaware of any previous evidence that bufenolides are to be found in the plant or animal kingdoms. An important conclusion of these experiments is that human beings (and presumably other mammals) possess a synthetic pathway to dihydropyronesubstituted steroids. F inal proof of their endogenous origin would require the demonstration of such a pathway. If the material described here is truly endogenous, questions remain as to its role in reproductive physiology and whether it is the only regulator of the sodium pump of man, rather than one of a family of such inhibitors. 23 T he assessment of its effects on sodium excretion and blood pressure in the intact animal will have to await the synthesis of larger quantities of the model compound. By analogy with previously synthesised similar compounds, we speculate that the compound will be inotropic, vasoconstrictive, and natriuretic. 24,25 Vol 348 • August 3, 1996 T his study was supported by the Special T rustees for St T homas’ H ospital and by M anchester M etropolitan U niversity. M ass spectrometry was carried out at the M ichael Barber C entre for M ass Spectrometry, U M IST . References 1 de Wardener H E, M ills IH , C lapham WF, H ayter C J. Studies on the efferent mechanism of the sodium diuresis which follows administration of intravenous saline in the dog. Clin S ci 1961; 21: 249–58. 2 Kramer H J, Backer A, Kruck F . Atinatriferic activity in human plasma following acute and chronic salt loading. K idney Int 1977; 13: 214–22. 3 G ruber KA, Whitaker JM , Buckalew VM . Endogenous digitalis-like substance in plasma of volume-expanded dogs. N ature 1980; 287: 743–45. 4 Edmondson RPS, T homas RD , H ilton PJ, et al. Abnormal leucocyte composition and sodium transport in essential hypertension. Lancet 1975; i: 1003–05. 5 Poston L, Sewell RB, Wilkinson SP, et al. Evidence for a circulating sodium transport inhibitor in essential hypertension. BM J 1981; 282: 847–49. 6 G ray H H , H ilton PJ, Richardson PJ. Effect of serum from patients with essential hypertension on sodium transport in normal leucocytes. Clin S ci 1986; 70: 583–86. 7 C ole C H , Balfe JW, Welt LG . Induction of a ouabain-sensitive AT Pase defect by uremic plasma. T rans A ssoc A m Phys 1968; 81: 213–20. 8 Edmondson RPS, H ilton PJ, Jones N F, et al. Leucocyte sodium transport in uraemia. Clin S ci 1975; 49: 213–16. 9 de Wardener H E, M acG regor G A. D ahl’s hypothesis that a saluretic substance may be responsible for a sustained rise in arterial pressure: its possible role in essential hypertension. K idney Int 1980; 18: 1–9. 10 Fagoo M , G odfraind T . Further characterization of cardiodigin, N a + ,K + -AT Pase inhibitor extracted from mammalian tissues. FEBS Lett 1985; 184: 150–54. 11 M organ K, Lewis M D , Spurlock G , et al. C haracterisation and partial purification of the sodium-potassium-AT Pase inhibitor released from cultured rat hypothalamic cells. J Biol Chem 1985; 260: 13595–600. 12 Lichtstein D , G ati I, Samuelov S, et al. Identification of digitalis-like compounds in human cataractous lenses. Eur J Biochem 1993; 216: 261–68. 13 G oto A, Yamada K, Ishii M , et al. Purification and characterization of human urine-derived digitalis-like factor. Biochem Biophys Res Commun 1988; 154: 847–53. 14 M athews WR, D uC harme D W, H amlyn JD , et al. M ass spectral characterization of an endogenous factor from human plasma. H ypertension 1991; 17: 930–35. 15 Lewis LK, Yandle T G , Lewis JG , et al. Ouabain is not detectable in human plasma. H ypertension 1994; 24: 49–55. 16 D oris PA, Jenkins LA, Stocco D M . Is ouabain an authentic mammalian substance derived from the adrenal? H ypertension 1994; 23: 632–38. 17 Laredo J, H amilton BP, H amlyn JM . Ouabain is secreted by bovine adrenocortical cells. Endocrinology 1994; 135: 794–97. 18 Bagrov AY, F edorova OV, D metrieva RI, French AW, Anderson D E. Plasma marinobufagenin-like and ouabain-like immunoreactivity during saline volume expansion in anesthetized dogs. Cardiovasc Res 1996; 207: 296–305. 19 M orris JF, M cEachern M D , Poston L, et al. Evidence for an inhibitor of leucocyte sodium transport in the serum of neonates. Clin S ci 1987; 73: 291–97. 20 H ilton PJ, Johnson VE, Jones RB, Patrick J. T he effects of alterations in the external sodium concentration on human leucocyte sodium and potassium transport in vitro. J Cell Physiol 1981; 109: 323–32. 21 de Bold AJ. Atrial natriuretic factor of the rat heart: studies on isolation and properties. Proc S oc Exp Biol M ed 1982; 170: 133–38. 22 T ymiak AA, N orman JA, Bolgar M , et al. Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus. Proc N atl A cad S ci US A 1993; 90: 8189–93. 23 H ilton PJ, H ilton M J, Forni LG . Poisons and regulators of the sodium pump. H ypertension 1995; 25: 460. 24 G litsch H G , Pusch H , Zylka C . T he action of 22,23-dihydrobufalin and other cardioactive steroids on contraction and active sodium/potassium transport of sheep cardiac Purkinje fibres. N aunynS chmiedebergs A rch Pharmacol 1990; 342: 598–604. 25 Stache U , H aede W, Radscheit K, Fitsch W. G er Offen 2,033,599. Chem A bs 1972; 76: 86021m. 305