between normal children and children with primary

Transcription

1 Arhives of Disease in Childhood, 1989, 64, odium transport in erythroytes: differenes between normal hildren and hildren with primary and seondary hypertension M UCHIYAMA, V HAH, C E DAMAN WILLEM, AND M J DILLON Renal Unit, Hospital for ik Children, Great Ormond treet, and Department of Paediatri Nephrology, Institute of Child Health, London UMMARY The following measurements were made in normal hildren, hildren with primary hypertension, and hildren with seondary hypertension: erythroyte intraellular sodium onentration, total sodium efflux rate onstant, and maximum binding of ouabain to erythroytes refleting the number of sodium/potassium adenosine triphosphatase pump sites Children with primary hypertension had a signifiantly higher mean erythroyte intraellular sodium onentration (8-2 ompared with 6-6 and 6*7 mmol/l ells), and signifiantly lower total sodium efflux rate onstant (571 ompared with and -6197) and maximum binding of ouabain to erythroytes (91 ompared with 11-7 and 11 nmol/l ells) than normal hildren and hildren with seondary hypertension, respetively ine the original report by Losse et al in 1961 that intraellular sodium was inreased in the red blood ells of patients with essential hypertension, various abnormalities of ion transport have been reported in patients with primary hypertension,24 and it has been postulated that essential hypertension may be aused by disturbane of membrane eletrolyte transport Beause it is diffiult to study the ells in the walls of blood vessels where abnormalities may affet blood pressure, irulating blood ells-for example, leuoytes and erythroytes-have been used to study the transport of eletrolytes aross membranes Few studies have been undertaken on transport of sodium aross ell membranes in hildren with hypertension5 6 We therefore undertook a study of erythroyte sodium transport in hildren with primary hypertension and ompared the findings with those in normal hildren and in patients with seondary hypertension ubjets and methods We studied 15 normal hildren who ated as ontrols (range 5-15 years), 12 hildren with primary hypertension (age range years), and 27 hildren with seondary hypertension (age range 5-15 years) The ontrol hildren, whose blood was being taken for another reason during the ourse of investigations of minor illnesses, did not have any family 224 history of hypertension Children with primary hypertension showed signifiant inreases in blood pressure (greater than the 95th entile for age) on repeated measurements and were not reeiving antihypertensive treatment No other auses for their hypertension were found on thorough examination All patients with seondary hypertension had renal hypertension, and in most of these the blood pressure was renin dependent All the hildren with primary hypertension had normal plasma renin ativities for their ages (less than 899 ng angiotensin I/l/hour or 69 ng angiotensin Ill/hour Eight of 27 hildren with seondary hypertension were reeiving treatment; six were reeiving propranolol and hydralazine, and two were reeiving aptopril The study was approved by the ethial ommittee of the Hospital for ik Children and the Institute of Child Health amples of 5 ml of fresh heparinised blood were taken; 2 ml of eah were used for measurement of the 22sodium efflux, and the remaining 3 ml were separated into plasma and erythroytes at 3 rpm The erythroyte intraellular sodium onentration was measured by flame photometry with IL 943 flame photometer (Instrumental Laboratory) after washing erythroytes three times with 155 mmol/l of old holine hloride (4 C) The 22sodium efflux rate onstant was measured by a modifiation of the method used by Fitzgibbon et al7; 4) [tci of 22 sodium (22 [tci/ml, Amersham International) was Arh Dis Child: first published as 11136/ad on 1 February 1989 Downloaded from on 6 Otober 218 by guest Proteted by opyright

2 added to 2 ml of whole blood and inubated at 37 C for one hour Labelled erythroytes were obtained and washed four times with old isotoni saline A sample of 25 Rl of paked erythroytes was added to 5 gl of their own plasma, and reinubated at 37 C for one hour; 25 pl of a ell suspension were taken into ounting tubes both before and after this inubation Finally, 25 tl of inubated blood was entrifuged at 12 rpm for three minutes (Eppendorf miroentrifuge) to obtain 125 RI of supernatant for ounting In eah tube 22sodium was ounted with a LKB gamma ounter (1282 Compugamma) The efflux rate onstant (Ke) was determined by using the following equation: ln (natural log) (1-N5/N)= -Ket, where NO=22sodium present/unit volume of suspension of inubating ells (mean of the ounts/ unit volume of suspension taken at time and 6 minutes), Nt=22sodium present per unit volume of supernatant at time 6 minutes The binding of ouabain to erythroytes was measured by a modifiation of the method used by Gardner and Conlon8 Paked erythroytes, whih were washed three times with old isotoni saline (4 C), were resuspended in trometamol (TRI) buffer (sodium hloride 13 mmol/l, surose 2 mmol/l, gluose 1 mmol/l, and TRI 1 mmol/l, ph 74) to a paked ell volume of -15 A stok solution of ouabain (1 mmol/l in 1% ethanol) was prepared and kept at 4 C A sample of 1 il of 3H ouabain (37 Ci/ml, Amersham International) was plaed into a glass tube and evaporated under nitrogen The residue was redissolved with 5,ul of isotoni saline, of whih 1 RI was diluted in 2*6 ml of isotoni saline on assay Two fold serial dilutions were then made four times Of the erythroyte suspension, 3 [tl were inubated with 5 [tl of the diluted radioative ouabain at eah dilution, and with 5 RI of isotoni saline or 1 mmol/l of old non-radioative ouabain at 37 C for two hours ubsequently the unbound ouabain was removed by washing three times with isotoni saline, and the final pellet resuspended in 2,ul of 1% trihloroaeti aid After entrifuging 1 RI of its supernatant was used for liquid sintillation ounting (LKB; 121 Ultrobeta) in 3 ml of Coktail T (British Drug Houses) Non-speifi binding was estimated by replaing 5 p1 of isotoni saline with 5 gl of 1 mmol/l of old ouabain The amount of ouabain speifially bound was alulated by subtrating the non-speifi ounts from the total ounts Maximum binding of ouabain to erythroytes was alulated using a urve fitting omputer programme All determinations were alulated in dupliate Intra-assay oeffiients of variation were 2-4%, odium transport in erythroytes %, and 6-2% for intraellular sodium onentration, total sodium efflux rate onstant, and maximum binding of ouabain to erythroytes, respetively Results were expressed as mean (EM) and analysed parametrially by the unpaired tudent's t test Results In the hildren with primary hypertension, mean erythroyte intraellular sodium onentration was signifiantly higher (8-2 (63) mmol/l ells) than in E E Ca) C E a C, CA m 14] , 3 2 i I * U 1 hildren primary seondary (n=15) hypertension hypertension (n=11) (n=24) Fig 1 Intraellular sodium onentration in erythroytes from normal hildren, hildren with primary hypertension, and hildren with seondary hypertension Arh Dis Child: first published as 11136/ad on 1 February 1989 Downloaded from on 6 Otober 218 by guest Proteted by opyright

3 226 Uhiyama, hah, Daman Willems, and Dillon C az x 4-1 H) E 12] 1 *111-1 I so v so hildren primary seondary (n= 15) hypertension hypertension (n=12) (n=27) Fig 2 Total sodium efflux rate onstants in erythroytes from normal hildren, hildren with primary hypertension, and hildren with seondary hypertension ontrol hildren (66 (32) mmol/l ells), and in hildren with seondary hypertension (67 (26) mmol/l ells) (p<5) (fig 1) In hildren with primary hypertension, mean total sodium efflux rate onstant was signifiantly lower (571 (-4)) than in ontrol hildren (6983 (4)), and in hildren with seondary hypertension (-6197 (-3)) (p<-1) (fig 2) The mean maximum binding of ouabain to erythroytes that reflets the number of pump reeptor sites was redued in hildren with primary hypertension (9-1 (-45) nmol/i ells) ompared with ontrols (11-7 (73) nmol/l ells), and with those with seondary hypertension (11- (-49) nmol/l ells) (p<-5) (fig 3) Disussion There are several pathways for ative transport of r sodium in erythroytes Firstly, there is sodium/ lithium ountertransport, whih is equivalent to sodium/sodium ountertransport This hannel is thought to exhange a single external sodium ion for a single internal one This annot affet the overall distribution of sodium aross ell membranes and there is, therefore, some doubt about its physiologial role9 Canessa et al,lo however, reported that ountertransport was inreased in erythroytes in patients with essential hypertension, suggesting that this system ould be a marker for essential hypertension The seond system that has been studied is frusemide sensitive sodium/potassium otransport This pathway lies dormant in the ell membrane unless intraellular sodium onentrations rise above a ritial point, and ats as a supporting mehanism for sodium extrusion Garay et al reported that the ativity of this system was dereased in erythroytes of patients with essential hyper- - o 4) CL m * I I* 3 hildren primary seondary (n=1 5) hypertension hypertension (n=1 2) (n=22) Fig 3 Binding of ouabain to erythroytes representing pump reeptor sites in normal hildren, hildren with primary hypertension, and hildren with seondary hypertension Arh Dis Child: first published as 11136/ad on 1 February 1989 Downloaded from on 6 Otober 218 by guest Proteted by opyright

4 tension, suggesting that this system ould also be a marker for essential hypertension'1 A third system is the sodium pump, whih is the best desribed and most widely studied pathway for ellular sodium homoeostasis The ativity of the pump system is onsistently low in white ells of adult patients with essential hypertension,12 13 but inonsistent results have been obtained in erythroytes that are thought to be aused by the ativity of the pump being muh slower in erythroytes than in white ells9 It was impossible to study white ells in hildren, however, beause more than 1 ml of blood would be required for eah assay, so we hose to study erythroytes by measuring erythroyte intraellular sodium onentration, total sodium efflux rate onstant and maximum binding of ouabain to erythroytes instead of sodium/lithium ounter-transport or sodium/potassium otransport Few studies have been undertaken on erythroyte sodium transport in hildren with primary hypertension Mongeau5 6 reported that hildren with labile hypertension tended to have dereased sodium/potassium otransport and those with stable hypertension inreased ountertransport Although interesting, however, these findings may not ontribute to our understanding of the mehanisms onerned in developing hypertension, beause the methods used for measuring sodium transport either reflet ativity of a system with little physiologial importane or rely on laboratory indued unphysiologial onditions In the present study eight hildren with seondary hypertension were reeiving antihypertensive drugs (aptopril, propranolol, or hydralazine) We did not find any differenes in erythroyte sodium transport between two groups of hildren reeiving or not reeiving hypotensive treatment, even though the drugs used have been reported to have some effet on sodium transport aross the ell membranes in adults14 The hildren who ated as ontrols were age mathed with the hildren with seondary hypertension, but the hildren with primary hypertension were a little older We have not, however, observed any differenes in erythroyte sodium transport assoiated with age in hildren after infany Differenes in age and mediation were therefore negligible among the three study groups Our findings that hildren with primary hypertension had signifiantly higher erythroyte intraellular sodium onentration and signifiantly lower total sodium efflux rate onstant and maximum binding of ouabain to erythroytes than ontrol hildren and hildren with seondary hypertension, onfirm results reported by workers studying adults with hypertension It seems likely that the high erythroyte intraellular sodium odium transport in erythroytes 227 onentration seen in hildren with primary hypertension is a result of low total sodium efflux rate onstant aused by the small number of sodium pump sites These findings suggest that measurements of erythroyte intraellular sodium onentration, total sodium efflux rate onstant, and maximum binding of ouabain to erythroytes may have some diagnosti value in positively identifying hildren with primary as opposed to seondary hypertension, and may have a preditive value in identifying hildren who are at risk of developing essential hypertension in adult life There is, however, substantial overlap among the groups of patients and further study is urrently being undertaken to establish whether fators suh as a family history of essential hypertension play a part in modifying transport of eletrolytes aross ell membranes It is not yet known whether the erythroyte sodium transport defet is aused by the hypertension, is a ause of the hypertension, or is just a marker9 We thank Dr H Bobinski of Manhester University, Dr PL Weissberg of the Queen Elizabeth Hospital, Birmingham, and Dr G agnella of the Charing Cross Hospital Medial hool, London, for their tehnial advie This work was suppported by the Kidney Researh Aid Fund, Charlotte Parkinson Researh Fund, ER quibb and ons Ltd, John Herring and Friends, and the Child Health Researh Appeal Trust Referenes 'Losse H, Wehmeyer H, Wessels F Wasser-und Elektrolytgehalt von Erythrozyten bei arterieller Hypertonie Klin Wohenshr 196;38: Tosteson DC, Adragna N, Bize I, olomon H, Canessa M Membranes, ions and hypertension Clin i 1981;61(suppl 7): 5-lOs 3Anonymous Cells, ions and blood pressure Lanet 1982;ii: Postnov YV, Orlov N Cell membrane alteration as a soure of primary hypertension J Hypertens 1984;2:1-6 5Mongeau JG Value of measurement of erythroyte ation fluxes as a geneti marker in essential hypertension of adolesents and hildren In: Brodehl J, Ehrih JHH, eds Paediatri nephrology Berlin: pringer-verlag, 1984: Mongeau JG Erythroyte ation fluxes in essential hypertension of hildren and adolesents Int J Pediatr Nephrol 1985;6:41-6 Fitzgibbon WR, Morgan TO, Myers JB Erythroyte 22Na efflux and urinary sodium exretion in essential hypertension Clin i 198;59:195s-7 8 Gardner JD, Conlon TP The effets of sodium and potassium on ouabain binding by human erythroytes J Gen Physiol 1972;6: Weissberg PL Ion transport studies-tehnial problems J Hypertens 1983;1 (suppl 2): Canessa M, Adragna N, olomon H, Connoly TM, Tosteson DC Inreased sodium-lithium ountertransport in red ells of patients with essential hypertension N Engl J Med 198;32:772-6 Garay RP, Elghozi JL, Dagher G, Meyer P Laboratory Arh Dis Child: first published as 11136/ad on 1 February 1989 Downloaded from on 6 Otober 218 by guest Proteted by opyright

5 228 Uhiyama, hah, Daman Willems, and Dillon distintion between essential and seondary hypertension by measurement of erythroyte ation fluxes N Engl J Med 198; 32: Edmondson RP, Thomas RD, Hilton PM, Patrik J, Jones NF Abnormal leuoyte omposition and sodium transport in essential hypertension Lanet 1975 ;i: Poston L, ewell RB, Wilkinson P, et al Evidene for a irulating sodium transport inhibitor in essential hypertension Br Med J 1981;282: Thomas RD, Edmondson RP, Hilton PJ, Jones NF Abnormal sodium transport in leukoytes from patients with essential hypertension and the effet of treatment Clinial iene and Moleular Mediine 1975;48:169s-7 Correspondene to Dr MJ Dillon, Renal Unit, Hospital for ik Children, Great Ormond treet, London WC1 3JH Aepted 11 August 1988 Arh Dis Child: first published as 11136/ad on 1 February 1989 Downloaded from on 6 Otober 218 by guest Proteted by opyright