EXTRACORPOREAL Extracorporeal circulation causes release of …downloads.hindawi.com/journals/mi/1999/648738.pdf · Extracorporeal circulation (ECC) is fundamental for cardiac surgery

Extracorporeal circulation causesrelease of neutrophilgelatinase-associated lipocalin(NGAL)

Per JÈonsson,1,CA Marie-Louise StŒhl2 andKjell Ohlsson2

1Department of Cardiothoracic Surgery, LundUniversity Hospital and 2Department of SurgicalPathophysiology, University Hospital MAS, MalmÈo,Sweden

CACorresponding AuthorTe l: (+46) 46 171683Fax : (+46) 46 158635

EX TRA CORPOREA L cir culation (ECC) used during cardiacsurgery causes activation of seve ral inflam m atorysystem s. These even ts are not fully understood butare re sponsible for com plications durin g the im m edi-ate pos toperative pe riod. Neutroph il ge latinase-asso-ciated lipocalin (NGAL), a m em ber of the ex pandinglipocalin fam ily, has r ecen tly been des cribed as anin flam m atory prote in . In this s tudy, th e release ofNGAL into th e cir culation in 41 patien ts undergoin ghear t surgery w ith ECC was evaluated. A 4- to 5-foldelevation of th e concentration of NGAL in plasm a wasobserved during the im m ediate postoperative coursew ith a rapid elim ination dur in g the firs t postoper-ative day. Four patients undergoin g lung surgery(w ithout ECC) were also s tudied. The plasm a concen -tration of NGAL only in creased w ith a factor of 1.1–2.2over th e operation . We conclude that NGAL is r eleasedin to th e cir culation durin g hear t surgery, probably asa re sult of th e in flam m atory activation of leukocyte sin itiated by the ex tracorporeal c irculation.

Key w ords: Cardiac surgery, Ex tracorporeal c irculation,Leukocyte ac tivation, Ne utrophil ge latinase -assoc iatedlipocalin

Introduction

Ex tracorpore al circulation (ECC) is fundamental forcardiac surge ry but it also induce s a syste mic inflam-matory re sponse w hich may cause morbidity andmortality.1 Various inflammatory cascade systems areactivated and e ffec ts are seen on coagulation andfibrinolysis . Ac tivation of neutrophil leukocytesoccurs and prote olytic and lysosomal enzymes arere leased, contributing to the incre ased vascularpermeability.2

Neutrophil ge latinase -assoc iated lipocalin (NGAL)is a member of the re cently desc ribed lipocalin familyand w as named afte r co-purific ation w ith leukocytegelatinase from sec retory granule s in human poly-morphonuclear leukocyte s.3 – 4 It has later also be engiven the name human neutrophil lipocalin, HNL.5

NGAL is a 24-kDa protein and ex ists in tw o mainforms, as a monomer and as a dimer.4 – 5 The functionof NGAL has ye t to be clarified. Production of NGALhas been demonstrated in in vitro inflammatorymode ls. Our purpose w as to e luc idate w hether NGALis re leased during ex tracorporeal c irculation.

Patients

Forty-one e lective patients undergoing elective heartsurge ry w ith ex tra-corporeal circulation w ere studiedregarding possible re lease of NGAL. The male /female

ratio w as 31/10 and the median age was 62 years(range , 36–79). The surgic al procedures includedcoronary bypass (32 patients), valve re placement(seven patients) and coronary bypass+valve replace-ment (two patients). The median time of ex tracorpor-eal circulation w as 83 min (range, 31–220) and themedian aortic cross-clamp time w as 50 min (range,14–118). The temperature during ex tracorporeal cir-culation w as 28°C.

Four patients unde rgoing major thorac ic surgeryw ithout the use of ECC were also studied. The sepatients had lung rese ctions via thoracotomy.

Methods

ECC patients

Blood sample s we re draw n off into EDTA-containingtubes preoperatively, 3 h afte r removal of the aorticclamp and on the first and second postope rative days.After centrifugation, plasma w as separated and storedat –30°C until analysed. Three samples we re missingfrom the se cond postoperative day.

Non-ECC patients

The corre sponding sample s w ere draw n but thesecond sample w as collected 3 h after closure of theskin.

ISSN 0962-9351 print/ISSN 1466-1861 online/99/030169-03 � 1999 Taylor & Francis Ltd 169

Short Communication

Mediators of Inflammation, 8, 169–171 (1999)

Laboratory analysis

The concentration of NGAL was determined by anenzyme-linked immunoassay.4

For the phagocytosis ex periment 2 ml humanse rum and 4 3 109 yeast particles w ere incubated for15 min at 37°C. The mix ture w as w ashed in 0.15 MNaCl, c entrifuged and re suspended to a volume of1 ml. The yeast was then incubated w ith 6.5 ml humanvenous blood in constant shaking for 30 min at 37°Cfollow ed by centrifugation at 300 3 g for 5 min. Atotal of 200 m l of the supe rnatant w as separated onSuperose 12 using an HPLC system (LKB). Two patientsamples , w ith high concentration of NGAL, w ere also

se lected for separation. The column w as run at a flowrate of 0.8 ml/min and e lution volume 0.5 ml. Thefractions were te ste d w ith ELISA for NGAL contentand measured in a spectrophotometer at 405 nm. Theidentification of the monomer and dimer w as madeusing Western blot as de scribed previously.4

Statistics

Median value s w ere calculated. Correlation betw eentime on cardiopulmonary bypass and levels of NGALw as te ste d using the Spearman rank correlation te st.Differences in immediate NGAL re lease betw een ECCand non-ECC patients w as calculated using the Mann–Whitne y U-te st.

The study w as approved by the local humaninvestigations committee at Lund Univers ity andinformed consent w as obtained.

Results

A 4- to 5-fold e levation of the plasma concentration ofNGAL w as demonstrated afte r the use of cardiopulmo-nary bypass (Fig. 1). The preoperative median concen-tration was 55 m g/l (range, 23–191). The max imumconcentration of NGAL, 234 m g/l (range , 82–574),w as observed 3 h after removal of the aortic clamp.The level of NGAL at this point corre lated to the timeon cardiopulmonary bypass (r=0.37; P=0.02). Themedian concentration of NGAL on postope rative day1 w as 101 m g/l (range, 36–637) and 84 m g/l (range,39–1011) on postope rative day 2. All patients but onehad lower leve ls of NGAL on postope rative days 1 and2 compared to the leve ls 3 h afte r removal of the

P. Jo n s s o n et al.

170 Mediators of Inflammation Vol 8 1999

FIG. 1. Median concentration of plasma-NGAL in samplesfrom patients operated with ECC ( d ) and without ECC ( s ).

FIG. 2. Immunoreactive NGAL (measured as absorbance at 405nm) after separation on HPLC Superose 12. Patient sample after3 h ( d ) and postoperative day 1 ( 3 ) and in vitro activation ( s ).

aortic cross-c lamp. The only ex ception w as a patientw ith postoperative intermittent bleeding requiring are-operation a few days later due to massive haemo-thorax . The pre operative concentration of NGAL inplasma in this patient w as 169 m g/l and the post-operative values we re 574, 637 and 1011 m g/l,respective ly.

Patients unde rgoing thoracotomy and lung surgeryhad less pronounced incre ase s in plasma NGALconcentration during the immediate postope rativecourse compared to patients operated w ith ECC(P<0.01). The 3-h leve ls we re increased, compared tothe pre operative levels, by a fac tor of 1.1, 1.3, 1.4 and2.2, respective ly.

After chromatography, immunoreactive NGAL e lu-te d in tw o peaks. The first and smaller peak correlatesto the monomer,4 w hile the majority of immunor-eactive NGAL w as found as a dimer (Fig. 2).

Discussion

A few reports have been presented demonstrating there lease of NGAL in inflammatory disease s. Levels ofplasma-NGAL w as e levated about 10 times in patientsw ith pe ritonitis. Very high leve ls , 37 mg/l, w e re foundin peritoneal ex udates.4 Local libe ration of NGAL hasbeen demonstrated in the gingiva during pe ridontitis6

and in colonic epithelium during dive rticulitis andappendicitis .7

Increased plasma leve ls of NGAL have also be enfound in patients w ith ischemic cerebrovasculardiseases .8 Ischemic cardiac disease does not seem tocause re lease of NGAL according to the normalpreoperative values in our se rie s w here the majorityof the patients included needed coronary re-vascular-isation. How ever, heart surge ry induces liberation ofNGAL into the circulation. This is probably due to theuse of ex tracorpore al c irculation, causing an inflam-

matory re sponse including the ac tivation of leuco-cytes since the patients unde rgoing major thorac icsurge ry w ithout ECC have a more limited initialre lease of NGAL.

NGAL w as circulating mainly in its dimer form.4 Thesignificance of this finding has to be furtherevaluated.

The turnove r of intravenously inje cted humanNGAL in rats is fast.4 In the present series , the peakplasma leve ls of NGAL, 3 h after removal of the aorticclamp, is more than halved on the first postope rativeday. Thus, NGAL could be a useful indicator of thecurrent degree of neutrophil ac tivation.

ACKNOWLEDGEMENTS. This study w as supported by grants from theMedical Faculty, Unive rsity of Lund, The Sw edish Medical Re search Council(Project. no. 03910) and the Sw edish Soc iety of Medicine.

References1. Edmunds LH Jr. Inflammatory response to c ardiopulmonary bypass. Ann

Tho rac Su rg 1998; 66: S12–S16.2. Jonsson P. Cardiopulmonary bypass c auses re lease of leukocyte protei-

nase-3. Sc a nd J Clin Lab Inve s t 1996; 56: 721–723.3. Kjeldsen L, Johnsen AH, Senglov H, Borregard N. Isolation and primary

structure of NGAL, a novel protein assoc iated w ith human neutrophilgelatinase . J Bio l Ch em 1993: 268: 10425 –10432.

4. Ax elsson L, Be rgenfeldt M, Ohlsson K. Studies of the re lease and turnoverof a human neutrophil lipocalin. Sca nd J Clin La b Inve s t 1995: 55:577–588.

5. Xu SY, Pe tersson CGB, Carlson M, Venge P. The development of an assayfor human neutrophil lipocalin (HNL)-to be used as a spe cific marke r ofneutrophil ac tivity in vivo and vitro. J Im m uno l Me tho ds 1994: 171:245–252.

6. Westerlund U, Ingman T, Lukinmaa PL e t a l. Human neutrophil gelatinaseand associated lipocalin in adult and localized juvenile peridontitis . J Den tRe s 1996; 75: 1553 –1563.

7. Nielsen BS, Borregaard N, Bundgaard JR, Timshe l S, Sehe sted M, KjeldsenL. Induction of NGAL synthe sis in epithelial ce lls of human colorectalneoplasia and inflammatory bow el diseases . Gut 1996; 38: 414–420.

8. Elneihoum AM, Falke P, Axelsson L, Lundberg E, Lindgarde F, Ohlsson K.Le ukocyte activation detec ted by increased plasma leve ls of inf lammatorymediators in patients w ith ischemic cerebrovascular dise ases . Stro ke1996; 27: 1734 –1738.

Received 11 February 1999;accepted in revised form 24 March 1999

NGAL a nd ex tra co rpo rea l c ircu la tio n

Mediators of Inflammation Vol 8 1999 171

Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

Documents

EXTRACORPOREAL Extracorporeal circulation causes release of …downloads.hindawi.com/journals/mi/1999/648738.pdf · Extracorporeal circulation (ECC) is fundamental for cardiac surgery