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Are Leukocytosis and Lipid Peroxidation Involved in Ischemic or Reperfusion Injury in Cardiac Surgery? M. A. Rashid and G. William-Olsson Departmem of Thoraeie and Cardiovascula r Surgery Sahlgrens ka Hosp ital. Gothenburg University. Gothen bu rg. Swede n

We have stud ied the hypothesis that free-radicaJ generatio n du ring ca rdiac surgery cou ld expla in pa rt ly th e pa th ophysi ology of ischemic or reperfusion Injury during ca rdio pulmona ry

bypass «(PB). Ten patients unde rgoing cardiac su rgery using ePB we re prcs pect ively studied. Malond ialdeh yde (MDA ) wa s

mea su red as a marker of fre e-radical-i ndu ced lipid per uxida tion ILPO) us ing th e thiobar bitu ric acid meth od . a nd leuk ocytes wer e cou nted du ring ischemia and rep erfusion . Both MOA a nd leuk ocytes incr ea sed significa ntly. especially a fter starting re perfusion. Th er e was significa nt cor relation between LPOa nd leuk ocytosis Ir - 0.8 , p < 0.005 ). It is concluded that free radi ca ls ge nera ted du ring ische mia a nd reperfusion lea d to a n increas c of LPO. wh ich is sho wn for the first time to bc assocrated with leukocy tos is in ca rdiac surgi cal pati cn ts. Th e implic ation of this obs ervat ion is of importa nce for th e tr eatme nt strategy using Ire e-radlcal scavenge rs in reducing the ha rm ful effects of ischemia and reperfusion in ca rd iac surgery. Key words Leukocytosis - Lipid per oxidation - Ischemic or reperfusion injury - Oxygen free rad icals - Cardia c s ur ge ry

Ist die Leukozyt ose und di e Llp idperux idat lon Illr d en Sc had en durch Isch ämie und Hepe r fuslon in d er Herzch iru rgie

mitverantworthr h? In der vorliege nde n Studi e u ntersuchte n wir d ie Hypoth ese. d aß d ie Bildung von freien Rad ikalen während herzchir urgischer EingrifTe wen igsten s zu ein em Teil die Pat hop hysiologie de r Ischämie und Reperfu sion wä h rend des CPB er klä re n ka nn . Dazu wu rden Patienten. die zu eine r ao rt o-korona re n Bypassopera tion a nsta nde n. pro sp ektiv u nters uch t. Malcndialdehyd (MOA). ein Marker der freien Rad ikale und de r von ihn en ver ursac hten Lipidpe rox idatio n (LPO). wurde mit Hilfe der Thio ba rbitursä ure methode wä h ren d der Ischä mie und Hep erfusion bestim mt. d ie Leu kozytenzahl a uch. MDA un d Leu kozyten zahl stiegen sign ifika nt vor allem nach Sta rt der Reperfusion, es besta nd ei ne Korrelation zwischen de r LPO u nd der l.cuko zvtose (r = 0 ,8, P < 0,005) . Somit ka n n bestäti gt werd en . daß die Bildun g von frei en Radika len wäh rend Ischäm ie und Heperfuslon zu eine r Erhöh ung der LPO führt und d ies mit ei ner Leukozytose bei herzchirurgisch en Patien ten verbu nden ist. Die Folge dieser Beoba chtung ist von gro ße r Bed eu tun g für d ie Behand lungsstrategie mit Radikalfän ge rn. um schä digende EfTektede r Ischä mie un d Heperfusion in der Her zch irurgie zu redu zier e n.

Introduct ion

Materials a nd ~I ethod s

lt has been suggested tha t oxygen free radi eals (OFR)gener ated during ea rdiopulmonary bypass (CPB) and the periods of isehemia a nd reper fusion du ring surgery eontribute to th e morb idity after eardiae surgery (I 0). Leukoeytes are one of the possib le sourees of OFR gene ra tion durin g eardiae su rgery (4) and have been implieated as a possible eause of reperfu sion inju ry in a nimals (9). One pathway by whieh OFR may indu ee taxie effects is through initiation of membrane lipid per oxidation (LPO) whieh alters membran e perrneability and may indu ee deleterious effects (3). Previous studi es have clearlv obse rved increa sed serum malondi aldehyd e (MDA) leveis in pati ent s with myoeardial ischemia

Ten patien ts we re electively operared upon using CPB for coro nary bypass gra fting surgery. The me an age wa s 66.7 ± 11.7 years (mea n ± SOl and ma lel fema le ratio was -+ : 1. All pat ients had good left-vent ricularfunction (eject ion fraction > 0.6). sin us rhyt hm . and no evide nce of other orga n diseas es a nd we re not tak lng corucoste roid the ra py. e PB was cond ucted using the sa me type of membrane oxyge na tor (Cobe Excel, Cobe Labo ra torfes. Inc. Lakewood . Colorade . USA) a nd primed with Ringerdex (Pha rmacia. Uppsala. Swede nl. A mo dified St. Th om as solutio n Il' ha rmacia. Uppsa la. Swede nl was use d to achleve ca rdio plcgta and gen er al hypoth ermia (28 °C) was instituted. Blood sa mpIes for assay of plasma MDA as a ma rker of the free radicals inducing LPO we re ta ken int o di potassium et hyle nediami noaceta te a nd s tor ed on ice befor e se pa ra tio n of plas ma at 2 000 g at 4 °C and othe r sampIes for mea su re ment of leukocytes were obta ined at the sta rt of CPB Ista rt of ische mial. at aorti c declamping Ista rt of re pe rfus ion) a nd at th e end of CPB, fro m th e a rterial line of the heart-lun g maehine . For determ inati on of MDA the blood sa mples we re centrifuged a nd a nalysed us ing the ßuorom etric ass ay of th e add uct formed with thi oharbitu ric actd as descr ibed by Yugi (12). LPO in pla sm a ts

(5 ).

The aim of this study was 10 investiga te wh ether leukocylosis occurring du ring ischemia and reperfusion leads to oxygen free rad ieal gene ration and lipid peroxidation and to find out if this explains pa rtly Ihe pathoph ysiology ofisehemie/ reperfusion injury in cardia c surgery during CPB.

Thorac. cardiovasc. Surgeon 39 (199 1)193- 195 © Georg Thieme Verlag Stuug an . New York

Received for Publi eation: J anuary 24. 199 1

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Su mma ry

M. A. Rashid und G. William-Olsson

Thorac. cardiovasc . Surgeon 3 9 (1991) 5

expressed by MDAInmoVml. The measurement of leukocyte count was perform ed using microcell counter ISysmex F-80 0 , Toa, Med , Elect, Camp, Ltd, Kube, Japan). The data w ere expresse d as mean± s tandard error ofthe mean (SEM), and were compared with the

paired-t-test. Correlation w as done using a simple regress ion ana lysis. A p value equal or less than 0 .0 5 is considered signific ant.

4

Results The re were no operative deaths . The mean extracorporeal circulation tim e was (109 ± 22 minu tes) and the aorticcross clamp ing time was (79 ± 21 minu tes).

3

MDA changes du ring ischemia and reperfu sion

,-lI-~~~~~~~~~-~~

o During CPB. the conce ntra tion ofplasm a MDArose progressively from 2.20 ± 1.3 nm ollml at the sta rt of CPB (start of ischem ia) to 3.19 ± 1.7 nmo llm l (p < 0.02) at aortic declam ping (start of reperfusion) and the n rose to 4.00 ± 1.2 nmollml at the end of CPB (p < 0.001 ) (Fig. I). The mean rate/m inute of plas ma MDA increased 10 .2 fold from 0.013 ± 0.01 8 nm ollmin during ischemia to 0.133 ± 0.177 nm ollmin durin g reper fusion (p < 0.01).

Corre/ation between /eukocgtosi s and MDA

80

1 20

1 00

Aortl c decla mping

e nd

ct

Minu tes

CPB

Fig. 1 Thechangesof malondialdehyde(M DA) concentrationnmol/ mlat the startof schemia (CPBstart),at thestartof reperfusion(aorticdeclamping)and atthe end ofCPB. Valu es are mean ± SEM. ' ''p < 0.001

ta ~

o

~

C ,

-Go

6

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...... LYMPH .... MONO+GRAN

o

U

' l-- - - - - - --r--::-

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20

ao

60

80

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CPB slart

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Fig.2 Thecount 01 white blood cells (WBCs), lymphocytes, and rnonocytes plus granulocytes (X109/ l) atthestartofischemia(CPB start), at thestartof repertusion (aortic declamping) andat theendofCPB.Values are mean ± SEM. " p < 0.01. '''p < 0.001

0.4

There wa s significant corre lation between MDA and leukocytosis durin g the early ph ase of ischemia (r ~ 0.8. P < 0.005) (Fig. 4).

0.3

Discuss ion

0.2

This study suggests that Or R may be a mechan ism in the path oph ysiology of CPB and lends strength to the clinical study of Ragston (10) who reported an increase in LPO produ cts afte r ao rtic declampi ng in pat ients undergoing myocar dial revasculari zation. OFR duri ng e PB could be de rived from the activat ed neu tro phils which releas e oxidants and pruteases, caus ing K· leak an d hemolysis (2). It is weil esta blished that e PB per se resulls in a complement mediated activat ion of polymorp honu clear leuk ocytes (PMNs) (I) . This activation corres ponds among other events to a produ ction of OFR. in particular, superoxide anions and hydrogen pero xide (7) . In ad dition , thes e activat ed PMNs release lactoferrin, a protein that binds iron in tbe extracel-

60

40

Start 01 CPB

Leukocgte changes during ischemia and reperfu sion The re was a significant increase in tbe circulating white blood cell count [x 10 9/L) which rose from 3.070 ± 1.067 at the start of ische mia to 4.620 ± 2.623 at the start ofreperfusion then to 10 .180 ± 2.917 (p < 0.001 ) at th e end of CPB (Fig. 2) and the rate of increas e was 15.1 times from 0.01 8 ± 0.036/m in during ischemia to 0.272 ± 0.271 /min during reperfusion (p < 0.00 1) (Fig. 3). Resp ective cha nges in the lym phocytes [x 109/L) were from 1.110 ± 0.37 to 1.38 ± 0.32 and the n to 2.26 ± 0.71 (p < 0.0 1) (Fig. 2) an d the rate of increase was from 0.007 ± 0.003/m in during ischemia to 0.182 ± O.I 77/min during reperfusion (26 times incr ease) (p < 0.00 1) (Fig. 3). Respective cha nges in the mono cytes plus gran ulocytes (x 10 9/L) fro m 2.09 ± 0.68 to 3.92 ± 2.73 and to 7.03 ± 1.63 (p < 0.01) (Fig. 2) and the rate of increa se was from 0.07 3 ± 0.174/ min during ische mia to 0.142 ± 0.24/m in during repe rfusion i.e . increased 1.9 times (p < 0.001) (Fig. 3).

20

LEUKO fRATE LYMPH fRATE MON+GRNfrat

0.1

o.oL--~ RE PERFUSION

IsCHEMIA 9

Fig.3 Therateofthecount (XI 0 / l )/ minuteoftheIeukocytes,lymphocytes, andmonocytesplusgranulocytes during ischemiaandreperfusion.Values are mean ± SEM . ' ''p < 0.001

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194

Thorac. cardiolJasc. S urgeofl39 (1991)

Are Leukocytosis and l.ipid Peroxidation lnro lced? y

5

= · .97 6.

+ 5.192,

Fig.4 The correlationbetween leukocytecount and MOAchanges during ischemia and repertusion Ir• 0.8. p < 0.005)

ä -sque ree : .649

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lular environme nt. It has been reported that th e sup ero xide anion ean mobilize lactoferrin-bound iron (6) thu s making free iron available for ealalyzing th e hydroxyl radi ealgenerating Haber-Weiss reaetion . This sehem e of events is likely 10 proee ed in a self-propagating manner beeause PMN-de rived free radi eals ean trigger the sec ret ion by mem brane phosp holipids of ehemotaetie substanees, eausing a fur ther reeruitment of aetivated PMNs, whieh in turn will prod uee additional free ra dieals (11). In our study , the positive eorrelalion between MDA and leukoeyte count suggests th aI as leukoeyle eounl inereases, probably OFR gen eration inerea ses as weil. MDA inereased during e PB notab ly 10.2 times du ring myoeardial reper fusion . The total leukocytes, Iymphoeytes and the concornitta nt gra nuloeytes plus monoeytes inereased by 15.1, 26 and 1.9 times resp ectively, However , the inerease of' leuk ocyt e count es pecially after reperfu sion may be allributed to OFR generanon espeeially su peroxide and the intermed iate hydrog en per oxide (2). Sinee the blood sampi es wer e ta ken from the arte rial line of th e heart-Iung maehin e, the ehanges in MOA and leukoeyte count a re probably not speeifie to Ihe heart. With this in mind . th e a brupt inerea se of MOA and leukocytosis immediately afte r starting reperfusion suggests that this is proba bly due to Ihe gen era tion and damaging eITeets of free radi eals, but Ihe souree ilself is still a maller of debale. However, a rece nt elinieal study (8) suggested a burs t of OFR at the start ofreper fusion ofa se leeled eoronary segment. In our study, th e pr olonged inerease of MOA following aorlie deelampin g suggests thaI OFR are prod ueed eontinously in greate r amounts upon reper fusion of the entire ischemic heart. Furt hermo re, th e presenee of ePB and blood trauma probably aeeentuates Ihe reperfus ion damage du e to OFR, whieh eould lead to a vieious eirele with myoeardial dysfunetion or delay in recovery . From the preeeding data , it is not un expected Ihatthis isehemiaJreperfusion sequenee results in production offree radi eals in th e myoeardium (13). Such a ph enom en on is also likely to oeeur du ring heart transpIantat ion wh en the isehemieally stor ed donor heart is abruptly reoxygenated by th e recepient's blood. Sinee myoeardial isehemia and reperfusion produee LPO due to OFR, such a production probably ought to be pr event ed, redu eed, or controlled in ca rdiac surgery in order to mak e this common lee hnique safer for Ihe patients ,

4 .5

4

5

Refer en ces I

c..

J . R. Plu th, 11. V. Schaff. T. A. Orszulak. 11. A . Hamburger. E. Solis. .\1. P. Kay, M . S. Claney, J. Kolif. a nd G. .\1.

Caea roccni. N.

Deeb. Com plem ent acüvatlon du ring CPR Compa riso n of bu bble and me mb ra ne oxygenato rs . J. Thorac . Cardiovasc. Surg. 91 (1986)

252-258 Chiu, D.• F. Kup ers, and B. Lubtn: Lipid peroxida tion in h um a n red cells. Semina rs in hem atolo gy 26 (1989) 257-276 3 Dohle. I.. K.. 11. G. Hili. a nd R. T. Holmo n. The thi oba rb itu ric acid reactio n and the auto xtdatio ns o f polyu nsa tura te d fatty acid m eth yl ester s. Arc hives Bioehern . Biophys . 98 (962) 253 .. Fantone. J. c.. and P. A. War d: Hole of oxygen- d erlved free radicals an d meta bolities in leukocyte-depe ndent inflamma tory reactions. Z

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Am. J. Path. 107 (198 21397 - 418 Fechner. J. . J. Gay . J . Y. Artigon. O. Bedu . J. Lee pe r. J. Eme rit. and Y. Grosgogeat: Study of membrane lipid peroxida üon in coro nary insufficiency. Press . med . 15 (1986) 1077 - 1080 Gauduel. Y.. and .\1. A. Dueelteroq: Role of cxygen radi cals in cardiac inju ry du e to reoxygenatlon. J. Mol. Cell. Card iol. 16 (198 41

459-4 iO l.ucchesi. B. H.• J. L. Romson, andS. R. Jolly: 0 0 leukocyt es inllue nce infarct size? In: Hearse. D. J .. and D. M. Yellon . eds.: The ra peutic a pproaches to myocardi al infarct size limit ati on . New York, Raven Pres s . 1984. pp 219-24 8 8 Roberts. M. J. D.. I. S. YOUrlg . T. G. Treu ton . E. R. Trimble. M. M. Khan. S. W. Ifebb. C. M . wttson. G. C. Pauerson. andA. A . J. A dge y : Transient re lease of lipid peroxides a fter coronary a rte ry ba lloon angioplasty. Laneet 336 (1990) 143- 145 '} Romson. J. L.. ß. G. Hook . S. L. Kunkel. G. D. Abrams. M. A. S chork. and 11. H. Lucchesi: Redu ction of the extent of isch cm ie myocard ia l inj ury by neutr oph il de pletion in th e dog. Circ . Res . 67 ( 983) 1016-1 023 10 ROyst ofl. 0. . J. S. Flem ing. J. B. Uesai. S. Wes t aby . and K. M. Tag/ ar: Incr eased pr odu ction of pe roxidatio n produ cts as sociate d with cardi ac ope rations . Evi dence for free ra dica l generatio n. J. Th orac. Card iovasc . Su rg. 91 (1986) 759 -766 11 Ward , P. A. G. O. Till, R. Kunkel. a nd C. Beauch am p : Evide nce for role of hydroxyl rad ical in compleme n t a nd neu trop hil dep end en t tiss ue injury. J. Clin. lnvest. 72 (1983) 789-801 IZ Yagi. K.: Assay for blood plasma or serum . Methods Enzymol. 105 (1984)328-33 1 13 Zw eier. J. L.. 1. T. Flaherly. and .\-1. L. Weisfedt: Direc t measur e ment of free radical generation following rep erfusion of ische mic myocardium. Proc . Natl. Aead . Sei. USA 84(1987) 1404-1408 1

J /oh eb A. Rashid. AI. D. Dep artm ent ofThoracic and Card iovasc ular Su rgery Sah lgrcnska hosp ita l. Got hen burg Uni versity S-4 13 45 Goth en burg Swede n

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