Rupture of abdominal aortic aneurysm (AAA)continues to result in significant morbidity and mor-tality.1 Since the initial reports of abdominal aorticaneurysm repair, there has been a steady and progres-

sive decline in the mortality reported for electivecases.2,3 Katz et al4 have noted a statewide trend forincreased numbers of elective AAA repair. However,the frequency of ruptured AAAs (RAAAs) has notdecreased and the mortality has remained unchanged.Population-based study results suggest that rupture ofAAA is associated with a 90% mortality, yet individualcenters report mortality rates that vary between 40%to 70%.5-9 Analysis of these clinical series has deter-mined many factors that contribute to the mortality inthese patients. Rapid operative intervention withouttechnical mishap is the current standard of therapy.10

Postoperative treatment is directed at correction offluid status, acid base imbalances, and hematologic

Ruptured abdominal aortic aneurysm, a “two-hit” ischemia/reperfusion injury:Evidence from an analysis of oxidativeproductsThomas F. Lindsay, MD, MSc, Xao P. Luo, PhD, Denis C. Lehotay, PhD,Barry B. Rubin, MD, PhD, Michael Anderson, MD, Paul M. Walker, MD,PhD, and Alex D. Romaschin, PhD, Toronto, Ontario, Canada

Purpose: Ruptured abdominal aortic aneurysm (RAAA) remains a lethal condition despiteimprovements in perioperative care. The consequences of RAAA are hypothesized to resultfrom a combination of two ischemia/reperfusion events: hemorrhagic shock and lowertorso ischemia. Ischemia/reperfusion results in tissue injury by diverse mechanisms, whichinclude oxygen free radical–mediated injury produced from activated neutrophils, xanthineoxidase, and mitochondria. Oxygen-free radicals attack membrane lipids, resulting inmembrane and subsequently cellular dysfunction that contributes to postoperative organinjury/failure. The purpose of this investigation was to quantify the oxidative injury thatoccurs as a result of the ischemia/reperfusion events in RAAAs and elective AAAs.Methods: Blood samples were taken from 22 patients for elective AAA repair and from 14patients for RAAA repair during the perioperative period. Plasma F2-isoprostanes wereextracted, purified, and measured with an enzyme immunoassay. Aldehydes and acyloinswere purified and quantified. Neutrophil oxidative burst was measured in response to areceptor independent stimulus (phorbol 12-myristate 13-acetate) with luminol-basedchemiluminescence.Results: Plasma from patients with RAAAs showed significantly elevated F2-isoprostanelevels on arrival at hospital and were significantly elevated as compared with the levels ofpatients for elective repair throughout the perioperative period (two-way analysis of vari-ance, P < .0001). Multiple regression showed a significant relationship between thephagocyte oxidative activity and F2-isoprostane levels (P < .013). Total acyloin levels weresignificantly higher in patients with RAAAs as compared with the levels in elective cases.Conclusion: The F2-isoprostane levels, specific markers of lipid peroxidation, showedthat patients with RAAAs had two phases of oxidative injury: before arrival at hospitaland after surgery. The significant relationship between the postoperative increases in F2-isoprostane levels and the neutrophil oxidant production implicates neutrophils in theoxidative injury that occurs after RAAA. New therapeutic interventions that attenuateneutrophil-mediated oxidant injury during reperfusion may decrease organ failure andultimately mortality in patients with RAAAs. (J Vasc Surg 1999;30:219-28.)

219

From the University of Toronto, Division of Vascular Surgery,Departments of Surgery and Clinical Biochemistry, TheToronto Hospital and The Hospital for Sick Children.

Supported by the Physicians of Ontario through PhysicianServices Incorporated.

Reprint requests: Thomas F. Lindsay, MD, 200 Elizabeth Street,Eaton 5-306, Toronto, Ontario, Canada M5G 2C4.

Copyright © 1999 by the Society for Vascular Surgery andInternational Society for Cardiovascular Surgery, NorthAmerican Chapter.

0741-5214/99/$8.00 + 0 24/1/98842

deficiencies. However, no specific treatments havebeen developed to decrease the mortality from thecardiovascular events or from the multiple organ fail-ure that frequently develops.11 Improvements in peri-operative care have not resulted in dramatic improve-ments in mortality.12

The principle difference between elective andRAAA repair is the period of hemorrhagic shock.Animal studies have established that hemorrhagicshock with resuscitation is a total body ischemia/reperfusion event.13 All aneurysm repairs experiencelower torso ischemia induced by aortic clamp applica-tion.14 Is it possible that these two ischemia reperfu-sion events act synergistically in patients with RAAAsto induce the organ failure and mortality observed?

Tissue or organ ischemia, which is prolonged andfollowed by reperfusion, will result in organ injuryand dysfunction.15 The severity of injury is depen-dent on many factors, which include the degree andduration of the ischemic insult and the sensitivity ofthe individual tissue to ischemia. During ischemia,the lack of oxygen leads to anaerobic metabolism anddepletion of energy stores, which may ultimately failto meet the tissue energy requirements. Once theenergy stores are depleted, membrane ion gradientsbegin to fail, membranes leak, cells swell, and theprocess of irreversible injury begins. Reperfusionrestores tissue oxygenation but initiates an inflamma-tory response that has been shown to cause furthertissue injury.16-18 The xanthine/xanthine oxidasesystem, activated neutrophils, and mitochondrialelectron transport chain leakage are documentedsources of activated oxygen species during reperfu-sion.19 These activated oxygen species and theirmetabolites (superoxide O2

–, hydrogen peroxideH2O2, hypochlorus acid HOCl, and hydroxyl radi-cals OH–) react with unsaturated fatty acids withinthe phospholipid bilayer of the cell membrane,resulting in lipid peroxidation. Oxidant-mediatedinjury is detected with measurement of lipid peroxi-dation products, such as malondialdehyde and otheraldehydes, lipid hydroperoxides, conjugated dienes,and acyloins. Cleavage of the long-chain fatty acidsafter lipid peroxidation results in fragmentationproducts, which add oxygen and result in the pro-duction of aldehydes. The spectrum of aldehydes ofvarious carbon chain lengths can be formed.However, they react rapidly and are quickly clearedfrom the circulation.20 Aldehydes are detoxified byseveral mechanisms, including one in which com-pounds called acyloins are formed by the pyruvatedehydrogenase complex, resulting in the addition ofa two-carbon fragment to saturated aldehydes.21

Recently, the F2-isoprostanes were discovered to bemarkers of oxidant injury.22 These prostaglandin-likecompounds are formed from free-radical catalyzedperoxidation of arachadonic acid through a non-cyclooxygenase enzyme pathway.22 These uniqueproducts can be measured and appear to be a sensi-tive and specific measure of free radical activity in sev-eral models of oxidant injury.23-25 Therefore, themeasurement of F2-isoprostanes, aldehydes, andacylions will permit an assessment of oxidative lipidperoxidation in patients with RAAAs.

The purpose of this investigation was to quanti-fy oxidative injury caused by the ischemia/reperfu-sion events in RAAAs and elective AAAs. Secondly, arelationship was sought between the recently identi-fied primed neutrophil oxidative burst of patients forboth elective repair and RAAA repair and the quan-tity of lipid oxidative products identified.

METHODSInformed consent for blood sampling was

obtained from patients for elective and RAAA repairin accordance with the Toronto Hospital andUniversity of Toronto Human Subject ResearchCommittee’s requirements. Blood samples were har-vested from the arterial line, central venous line, ora peripheral vein according to a preset sampling pro-tocol previously described.26 Blood samples weretaken: (1) before induction of anesthesia, (2) beforecross-clamp application, (3) before clamp removal,and (4) at 15, 60, and 240 minutes after clampremoval. Further samples (samples 1 to 7) weretaken on a daily basis. Blood was harvested into 7-mL sterile lavender vacutainer tubes that contained10.5 mg ethylenediamine tetraacetic acid (EDTA)and 0.014 mg potassium sorbate in 0.07 mL of 15%EDTA solution (Becton Dickinson, Franklin Lakes,NJ). The samples were kept at room temperatureand were rapidly transported to the laboratory foranalysis. The samples were gently rocked (BloodRocker Model R4185-10, Baxter, Deerfield, Ill) forseveral minutes before an aliquot was removed foranalysis of whole blood chemiluminescence and anautomated complete differential blood count. Theblood then was rapidly cooled to 4°C and cen-trifuged at 1800g for 15 minutes. The plasma wasdecanted into polypropylene tubes, snap frozen, andstored at –70oC. The aliquot of whole blood forchemiluminescence was measured at 37oC in anAutoluminat LB953 Luminometer with reagentsfrom ExOxEmis Inc (Little Rock, Ark). Each assaywas performed in triplicate with whole blood dilut-ed 1/1000 with buffer and 7.1 µmol/L phorbol 12-

JOURNAL OF VASCULAR SURGERY220 Lindsay et al August 1999

myristate 13-acetate (PMA) added to the luminol.Chemiluminescence was measured for a 30-minuteperiod, and the area under the curve was integrated.

F2-isoprostane measurement. Frozen plasma(0.5 mL) was thawed, and 5000 disintegrations perminute (dpm) of an internal standard (tritium labeledPGF2α) was added to quantify recovery during purifi-cation. Plasma protein were precipitated with ethanoland removed with centrifugation. F2-isoprostanes andall other fatty acid side chains were removed from theglycerol backbone by KOH hydrolysis at 40oC. ThepH was adjusted to less than 4 with HCl, and the solu-tion was loaded onto an activated reverse phase C18Sep-Pak Column (Waters Inc, Mississauga, Ontario).The column was washed with Ultrapure water(Millipore, Mississauga, Ontario) and hexane. F2-isoprostanes were eluted with ethyl acetate:methanol(99:1), evaporated to dryness under dry nitrogen, andstored under nitrogen at –70oC until further analysis.The samples were analyzed in duplicate for F2-iso-prostane content with a commercially availableenzyme-linked 8-isoprostane immunoassay (CaymanChemical Co, Ann Arbor, Mich). Recovery from thepurification step was analyzed with scintillation count-ing of the extract with Aquasol (Canberra-PackardInstruments, Meridan, Conn). The recovery range wasalways greater than 80%, and this was factored into thecalculation. F2-isoprostanes values are expressed aspg/mL of plasma.

Aldehyde and acylion measurement. The alde-hydes and acyloins in a 100-µL plasma sample werederivitized with O-2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride and incubated at roomtemperature for 30 minutes. The protein was precip-itated with methanol, and hexane was added fol-lowed by acidification with concentrated sulphuricacid. After centrifugation at 3000 rpm, the hexanelayer was removed, dried over sodium sulphate, andevaporated under nitrogen. The N,O-bis(trimethylsi-lyl)trifluoroacetamide reagent was added followed byincubation at 80oC to form trimethylsilyl ethers. A 1-µL sample was injected into the gas chromatographymass spectrometry (GC-MS).

Capillary action column gas chromatography withnegative ion chemical ionization mass spectrometry(GC-NICIMS) and ammonia as the reagent gas was used to analyze the pentafluorobenzyl-oxine-trimethylsilyl derivatives. This was performed in ahigh-sensitivity, research-grade quadrupole mass spec-trometer (VG-Trio 2A) with an interface to a HewlettPackard 5890 series II gas chromatograph (Miss-issauga, Ontario) equipped with a 30-m, 0.25-µm,DB-5 column (S & W Scientific, Folsom, Calif). The

temperature of the injection port was at 250oC, andthe column temperature was initially set at 60oC for 1minute, was increased by 20oC per minute until85oC, and then was raised 8oC per minute to 310oC.A specific ion for each derivative was chosen for select-ed-ion recording. The detection limits were between50 and 100 fmol per 1 µL injected. Concentrations inthe biologic samples were calculated from the ratio ofpeak height as compared with pure aldehydes and acy-loin standards that had been injected. Results areexpressed in nmol/L of plasma. This method is sensi-tive and completely specific for aldehyde and acyloinanalysis caused by the precise identification affordedby the GC-NICIMS technology.

In vitro production of F2-isoprostanes in acti-vated neutrophils. Neutrophils were purified fromhealthy human volunteers with a percoll-hypaquegradient and differential centrifugation. The neu-trophils were 95% pure by differential counting and98% viable by trypan blue exclusion. After Coultercounter determination of the neutrophil concentra-tion, cells were diluted to 1 × 106/mL. The neu-trophils were added to an equal volume of 10%platelet-poor autologous plasma and incubated withor without PMA (100 nmol/L) for 30 minutes.PMA is a phorbol ester that stimulates neutrophiloxidative burst by diffusing across the cell mem-brane and directly stimulates protein kinase C with-out necessitating membrane receptors. The sampleswere rapidly cooled to 4oC and centrifuged to pre-cipitate the neutrophils. The supernatant and neu-trophils were extracted for F2-isoprostanes contentas previously described and were analyzed with theimmunoassay. All the experiments were performedin duplicate, and the results were averaged. Theresults are expressed as pg F2-isoprostanes/1 × 106

neutrophils and per 2 mL of supernatant.Statistical analysis. Data were analyzed with the

SAS (SAS Institute, Inc, Cary, NC) for PC statisticalprogram, with Sigma Stat (SPSS Inc, Chicago, Ill), orwith both. Continuous data were compared with uni-variate t test analysis. The main effects of group (elec-tive vs RAAA), time (time course of measurementsduring and after AAA repair), and their interaction(group*time) were evaluated with two-way analysis ofvariance (ANOVA) for the dependent variables: F2-isoprostanes, and neutrophil oxidant burst measuredwith chemiluminescence. The relationship betweenchemiluminescence and F2-isoprostanes, time andgroup, was also evaluated with an analysis of covari-ance. A multiple linear regression with forward selec-tion combined with backward elimination was per-formed to identify the independent predictors of F2-

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isoprostane levels. For measures that were repeatedover the time course of AAA repair, a one-way repeat-ed measures ANOVA (RM-ANOVA) was used withcomparison to the pre-induction sample (PI) withDunnett test to correct for multiple comparisons.

The results are presented as means ± standarderrors in the text, tables,and figures. The resultswere considered statistically significant if the P valuewas less than .05.

RESULTSThe lower torso ischemia/reperfusion necessitat-

ed for elective AAA repair did not result in any sig-nificant alteration in plasma F2-isoprostane levels inthe perioperative period. The levels of this marker ofoxidant injury in these patients for elective AAArepair were slightly higher than the range reportedfor control groups in the literature (pre-induction56.0 ± 8.9 pg/mL compared with literature normals35 ± 6 pg/mL; Fig 1). This is a relatively small dif-

ference given the differences in measurement tech-nique (radioimmunoassay vs mass spectrometry) andthat our sample was of patients who underwent elec-tive aneurysm surgery. In contrast, in patients withRAAAs, the levels of F2-isoprostane were elevatedbefore surgical intervention 109.5 ± 16.5 pg/mL ascompared with elective 56.0 ± 8.9 pg/mL (P < .01,with t test). The levels in patients with RAAAsremained significantly elevated through the periop-erative period, and further significant increases werenoted on the third and fifth days after surgery in theRAAAs as compared with the preclamp release sam-ple (109.6 ± 46.5 pg/mL vs 154.4 ± 20.3 pg/mL;P < .05, with RM-ANOVA, and P < .05, withDunnett test; day 5). A two-way ANOVA on thecombined elective and RAAA repair F2-isoprostanedata set identified that both group and time weresignificant main effects. The interaction term(group*time) was also significant (P < .016), whichidentifies a significantly different rate of change in

JOURNAL OF VASCULAR SURGERY222 Lindsay et al August 1999

Fig 1. Plasma F2-isoprostanes in elective and ruptured abdominal aortic aneurysm repair.Plasma levels of F2-isoprostane measured during perioperative period for elective and rupturedaneurysms at indicated time points. Two-way analysis of variance showed significant group,time, and group*time effects.PI, Pre induction of anesthesia; PR, pre release of aortic clamp; 15 MIN, 15 minutes afterclamp removal; 1 HR, 1 hour after clamp removal; D1, first postoperative day; D3, third post-operative day; D5, fifth postoperative day; EAAA, elective abdominal aortic aneurysm repair;RAAA, ruptured abdominal aortic aneurysm repair. Repeated measures analysis of variance forboth groups, P < .001.

the F2-isoprostane levels between the two groups.An analysis of covariance with the natural log trans-formation of the F2-isoprostane levels revealed a sig-nificant group and time interaction (P < .001),which is further statistical evidence that the slopes,or the rate of change between the two groups overtime, was different (R2 = 0.165; P = .001). We con-firmed the ANOVA result with a multiple linearregression, which showed a significant associationbetween neutrophil oxidative burst (chemilumines-cence PMA), group, time, and the natural log of F2-isoprostane (R2 = 0.32; P = .013; Fig 2; oxidant pro-duction by neutrophils from patients with AAAs waspreviously published). No significant relationshipwas noted within each group (RAAA: R2 = 0.001; P= .71; EAAA: R2 = 0.004; P = .36).

Aldehyde and acylion measurements. Theentire spectrum of aldehydes and acyloins was mea-sured in both groups of patients with aneurysm. Nodifferences in aldehyde levels between the twogroups were noted (PI level, EAAA: 125.0 ± 6.4pg/mL vs RAAA: 138.9 ± 17.1 pg/mL; at 15 min-

utes, 124.5 ± 8.05 vs 142.5 ± 18.6; at 1 hour, 116.2± 7.4 vs 117.2 ± 7.6). Fig 3 shows the alterations inthe sum of all the acyloins (aldehyde detoxificationproducts) in both groups during the early postoper-ative period, which were measured with GC-NICIMS. The group of elective AAA repair shows aslow rise in the total acyloin content, which becamesignificant on the first postoperative day (RM-ANOVA, P < .0002; Dunnett test, P < .05, day 1 vsPI). In the RAAAs, the rise in acyloin level wasapparent within 15 minutes of clamp release, butthis was not statistically significant. By the first post-operative day, a near doubling of the total plasmaacyloins was noted in the RAAA group, which wassignificant, as compared with the preinduction levels(RM-ANOVA, P < .005; Dunnett test, P < .05, day1 vs PI). The patients with RAAAs had significantlyhigher levels of total plasma acyloin content as com-pared with the patients for elective repair both at 1-hour post–aortic clamp removal and at the first post-operative day.

The acyloin, 3-hydroxynonan-2-one, is derived

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Fig 2. Relationship between stimulated neutrophil oxidative burst and index of oxidant injuryover time. Linear relationship between stimulated neutrophil oxidative burst (chemilumines-cence phorbol 12-myristate 13-acetate [PMA]) and level of F2-isoprostanes measured in plasmaat that time point for patients for both elective and ruptured abdominal aortic aneurysm repair.Regression line calculated for entire data is F2-isoprostane level = 40.536 + 0.0042498 (PMAstimulated chemiluminescence). Regression line and slope for each group was significantly dif-ferent. However, overall line is shown to simplify the figure. Numbers beside each data pointrepresent time at which each sample was taken (samples 1 to 6 were taken on day of operation,and samples 7 to 11 were taken daily for first 5 postoperative days). Each point is mean of F2-isoprostane level plotted against neutrophil oxidant burst measured by chemiluminescence.

from the fusion of heptanal and pyruvate and showsa pattern of oxidative injury characterized by a dif-ference noted before surgery and further progressiveincreases, which become significant 1 hour after aor-tic clamp removal (Fig 4). This acyloin (which is oneof the acyloins included in the data represented inFig 3) slowly increases in the elective AAAs, with sig-nificant increases noted at 1 hour and on the firstpostoperative day as compared with the preinduc-tion level. The patients with RAAAs showed signifi-cant elevations of 3-hydroxynonan-2-one as com-pared with the patients for elective surgery at all timepoints measured (P < .01, RAAA vs EAAA).

In vitro production of F2-isoprostanes byactivated neutrophils. Unstimulated and stimulat-ed neutrophils were tested in vitro to determinewhether activated neutrophils resulted in increasedquantities of F2-isoprostane being produced.Neutrophils stimulated with PMA for 30 minutessignificantly increased the total content of F2-iso-prostane (unstimulated 103.6 ± 4.9 pg/mL vs PMAstimulated 146.7 ± 10.3 pg/mL, P = .001; Table I).The increase in F2-isoprostanes content was mainlyin the neutrophil fraction of the stimulated cells(unstimulated 53.2 ± 4.3 vs PMA stimulated 78.8 ±

8.1; P = .01). The rise in the F2-isoprostane contentof the supernatant failed to reach significance (50.4± 2.6 vs 67.8 ± 7.3; P = .14).

DISCUSSIONThis study examined two independent indices of

lipid peroxidation, F2-isoprostanes and acyloins/alde-hydes, in the plasma of patients for elective and RAAArepair. Alterations in the circulating levels of theselipid peroxidation products are indices of free-radicalgeneration and tissue injury.27 Free radicals are gener-ated at increased rates during reperfusion afterischemic events. Thus, identification of increases incirculating levels of lipid peroxidation productsreflects the magnitude of systemic free-radical tissueinjury that develops after ischemic events.

A reliable method to detect and quantify lipidperoxidation for in vitro and in vivo use has longbeen sought. Early measurements of lipid peroxida-tion were directed at malondialdehyde quantifica-tion via measurement of thiobarbituric acid reactivesubstances. Malondialdehyde measurements werefound to lack sensitivity and specificity, and thusresearchers directed their efforts at the measurementof intermediates of oxidative injury that were much

JOURNAL OF VASCULAR SURGERY224 Lindsay et al August 1999

Fig 3. Total plasma acyloins. Total acyloin content of plasma is represented. Repeated mea-sures analysis of variance for both groups, P < .001.*P < .05 vs pre-induction sample value.+P < .05 elective abdominal aortic aneurysm repair vs ruptured abdominal aortic aneurysmrepair.

more proximal in the chain reaction of events.Elevated conjugated dienes were measured in skele-tal and cardiac muscle after ischemia/reperfusioninjury that provided evidence of lipid peroxidation.However, they are technically difficult to mea-sure.28,29 Aldehydes are formed by lipid peroxida-tion and cleavage from fatty acids within cellularmembranes. Recently, a method to measure all alde-hydes and acyloins has been described.20 Severalgroups have measured aldehyde-protein fusionproducts to identify and quantify peroxidation injuryafter ischemia/reperfusion.21 Acyloins are derivedfrom the addition of pyruvate to a reactive aldehydevia the pyruvate dehydrogenase complex. The hearthas been shown to be a potential site of this reaction.

The measurement of F2-isoprostanes is the mostrecent development in the lipid peroxidation field.27

These products result from free radical attack onarachadonic acid.25 This results in the formation ofa stable five-member ring with striking similarity tothe prostaglandin F2α family. These compoundswere initially detected and quantified with GC-MS,with detection of the negative ion chemical ioniza-tion products. The immunoassay used in this studyhas been shown to correlate well with the results ofthe GC-MS analysis. Investigations have determined

that only minimal amounts of F2-isoprostane areproduced via a cyclooxygenase pathway and that thevast majority are caused by free-radical injury.30,31 Ina model of carbon tetrachloride–induced free-radicalinjury, F2-isoprostanes were detected initially in themembranes of liver tissue.32 This was followed by aprogressive release of F2-isoprostanes into the plas-ma, where their half life was found to be 16 minutes.Recently, increased levels of F2-isoprostane havebeen identified in patients with acute myocardialinfarction treated with thrombolytic therapy.33

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Fig 4. 3-Hydroxynona-2-one plasma levels. Repeated measures analysis of variance for bothgroups, P < .001.*P < .05 vs pre-induction sample value.+P < .05 elective abdominal aortic aneurysm repair vs ruptured abdominal aortic aneurysmrepair.

Table I. The F2-isoprostane levels from the super-natant, human neutrophil, and total content afterin vitro activation of oxidant burst with PMA at 30 minutes

Unstimulated PMA Stimulated

Supernatant 50.4 ± 2.6 67.8 ± 7.3Neutrophil 53.2 ± 4.3 78.8 ± 8.1 *Total 103.6 ± 4.9 146.7 ± 10.3 +

PMA, Phorbol 12-myristate 13-acetate.*P = .01 compared with unstimulated.+P = .001 compared with unstimulated.

Animal experiments with myocardial ischemia havealso shown similar results.34 These and other exper-iments have established F2-isoprostane as the cur-rent gold standard for measurement of oxidative tis-sue injury.

Diverse mechanisms and mediators that includeneutrophils, the xanthine/xanthine oxidase enzymesystem, and mitochondrial electron transport leakagecause oxidant-mediated tissue injury after ischemiareperfusion.35 Ischemia with or without reperfusion isa central mechanism of local and remote tissue injuryin many human diseases, which include myocardialinfarction, stroke, embolic vascular organ or limbocclusions, trauma, and hemorrhagic shock. Thedetection of lipid peroxidation products implicates anoxygen-free radical mechanism of injury.

The F2-isoprostane results show, for the firsttime, that the individual events that comprise repairof an RAAA (shock and lower torso ischemia) eachresult in significant elevations of lipid peroxidationproducts. The elevated levels of F2-isoprostane notedbefore surgical repair in the RAAAs identifies that the“first hit” of the oxidative injury had begun beforearrival at the hospital. The further significant increas-es noted after surgery appear to be caused by thecombination of hypotension and lower torsoischemia. We noted that RAAA repair, which necessi-tates lower torso ischemia, induces a second phase ofsignificant elevations in postoperative F2-isoprostaneslevels. This second rise in F2-isoprostanes is noted onthird and fifth postoperative days and is evidence ofpostoperative oxidative injury—“a second hit.”When our results are compared with single-hitevents, these injuries have a monophasic elevation inF2-isoprostanes.33,34 In studies of patients withmyocardial ischemia, the urinary metabolites of F2-isoprostane became elevated and fell rapidly.33,34

Animal studies also show rapid clearance of F2-iso-prostanes from the circulation after a single oxidativeevent.33 In elective AAA repairs, lower torso ischemiaalone did not result in a rise in F2-isoprostanes. Thus,the significant postoperative increases in the RAAAgroup suggest ongoing postoperative oxidativeinjury and thus provide evidence of a “two-hit”injury. Measurements of plasma acyloins, a secondspecific marker of lipid peroxidation, also show alarger degree of oxidative injury in the patients withRAAAs as compared with the elective repair group.Alterations in aldehyde levels may have been so tran-sient that measurement of differences betweengroups was precluded because of rapid detoxification.

The significant relationship between stimulatedneutrophil oxidant activity and the postoperative

levels of F2-isoprostane suggest a significant mecha-nistic link between these two events (Fig 2).Neutrophils are critical mediators of tissue injuryafter ischemia reperfusion events. However, they areby no means the exclusive cause of lipid peroxidationinjury.35,36 Although other mechanisms of oxidativeinjury are clearly operational, stimulated neutrophiloxidative burst is likely a significant mediator of thisinjury. The enhanced production of F2-isoprostanein vitro by stimulated neutrophils shows that theoxidative burst of these cells can be responsible forproduction of these lipid peroxidation products.These in vitro data lend support to the mathematicrelationship that we have demonstrated between thestimulated neutrophil oxidative burst and the plasmalevel of F2-isoprostanes.

Overall, the oxidative injury caused by lowertorso ischemia/reperfusion in patients for electiverepair is modest. The elevation in postoperative acy-loin levels suggests that an increase in lipid peroxi-dation develops early in the postoperative phase ofelective AAA repair. This level of oxidative injuryappears to be insufficient to increase F2-isoprostanelevels. This is in keeping with the low incidences oforgan failure and mortality that follow elective AAArepair.37

Recently, hemorrhagic shock and resuscitationhave been recognized as a whole body ischemia/reperfusion type injury.13 A dramatic reduction inorgan injury and mortality was observed when anantibody against the neutrophil CD18 adhesionreceptor, which prevents firm neutrophil adhesion,was administered during resuscitation in a model ofhemorrhagic shock.38 During resuscitation fromhemorrhagic shock, the no-reflow phenomenon hasbeen documented and neutrophil adhesion in thecapillaries has been described.39 Taken together, thesestudies support the hypothesis that shock and resusci-tation causes neutrophil activation. Also, prolongedlower torso ischemia by itself has been documented toresult in local and remote organ injury.14,40 Ourresults support the conclusion that each of these indi-vidual ischemia/reperfusion events results in oxidativetissue injury after repair of RAAA. The combinedinsult results in a systemic oxidative injury that chal-lenges the function of every organ. Our observationssuggest that therapeutic interventions directed at theoxidative injury, even if provided after the period ofhypotension during the operative repair of an RAAA,may prevent further tissue injury.

In summary, this study has identified two phasesof oxidative ischemia/reperfusion injury in thosepatients who undergo repair of RAAA with two spe-

JOURNAL OF VASCULAR SURGERY226 Lindsay et al August 1999

cific and sensitive markers of lipid peroxidation. TheF2-isoprostane results suggest a “two-hit” model ofinjury induced by hemorrhagic shock followed bylower torso ischemia in patients with RAAAs.Neutrophils have been shown to be activated byboth phases of RAAA.26 The relationship betweenthe F2-isoprostane levels and the oxidant productionactivity of the patient’s neutrophils suggests a rolefor neutrophil-induced oxidative tissue injury afterRAAA. Treatments directed at preventing neu-trophil activation or adhesion, or the scavenging ofactivated oxygen species, may reduce organ injuryand mortality after RAAA repair.

REFERENCES

1. Chen JC, Hildebrand HD, Salvian AJ, Hsiang YN, TaylorDC. Progress in abdominal aortic aneurysm surgery: fourdecades of experience at a teaching center. Cardiovasc Surg1997;5:150-6.

2. Johnston KW, Canadian Society for Vascular Surgery AneurysmStudy Group. Influence of sex on the results of abdominal aor-tic aneurysm repair. J Vasc Surg 1994;20:914-26.

3. Ernst CB. Abdominal aortic aneurysm [see comments]. NEngl J Med 1993;328:1167-72.

4. Katz DJ, Stanley JC, Zelenock GB. Operative mortality ratesfor intact and ruptured abdominal aortic aneurysms inMichigan: an eleven-year statewide experience. J Vasc Surg1994;19:804-17.

5. Bengtsson H, Bergqvist D. Ruptured abdominal aorticaneurysm: a population-based study. J Vasc Surg 1993;18:74-80.

6. Koskas F, Kieffer E. Surgery for ruptured abdominal aorticaneurysm: early and late results of a prospective study by theAURC in 1989. Ann Vasc Surg 1997;11:90-9.

7. AbuRahma AF, Woodruff BA, Lucente FC, Stuart SP,Boland JP. Factors affecting survival of patients with rupturedabdominal aortic aneurysm in a West Virginia community.Surg Gynecol Obstet 1991;172:377-82.

8. Akkersdijk GJ, van der Graaf Y, van Bockel JH, de Vries AC,Eikelboom BC. Mortality rates associated with operativetreatment of infrarenal abdominal aortic aneurysm in TheNetherlands. Br J Surg 1994;81:706-9.

9. Johansen K, Kohler TR, Nicholls SC, Zierler RE, ClowesAW, Kazmers A. Ruptured abdominal aortic aneurysm: theHarborview experience. J Vasc Surg 1991;13:240-7.

10. Bauer EP, Redaelli C, von Segesser LK, Turina MI. Rupturedabdominal aortic aneurysms: predictors for early complica-tions and death. Surgery 1993;114:31-5.

11. Maziak DE, Lindsay TF, Marshall JC, Walker PM. Theimpact of multiple organ dysfunction on mortality followingruptured abdominal aortic aneurysm repair. Ann Vasc Surg1998;12:93-100.

12. Lindsay TF, Johnston KW. Advances in vascular surgery. In:Whittemore AD, editor. Ruptured abdominal aortic aneurysm.From diagnosis to discharge. Mosby, Inc: St Louis, MO; 1995.p. 127-48.

13. Vedder NB, Winn RK, Rice CL, Chi EY, Arfors KE, HarlanJM. A monoclonal antibody to the adherence-promotingleukocyte glycoprotein, CD18, reduces organ injury andimproves survival from hemorrhagic shock and resuscitationin rabbits. J Clin Invest 1988;81:939-44.

14. Welbourn R, Goldman G, Kobzik L, Paterson IS, Valeri CR,Shepro D, et al. Role of neutrophil adherence receptors (CD18) in lung permeability following lower torso ischemia. CircRes 1992;71:82-6.

15. Ar’Rajab A, Dawidson I, Fabia R. Reperfusion injury. NewHorizons 1996;4:224-34.

16. Hernandez LA, Grisham MB, Twohig B, Arfors KE, HarlanJM, Granger DN. Role of neutrophils in ischemia-reperfu-sion-induced microvascular injury. Am J Physiol 1987;253(3Pt 2):H699-703.

17. Hill J, Lindsay TF, Ortiz F, Yeh CG, Hechtman HB, MooreFD Jr. Soluble complement receptor type 1 ameliorates thelocal and remote organ injury after intestinal ischemia-reper-fusion in the rat. J Immunol 1992;149:1723-8.

18. Granger DN. Role of xanthine oxidase and granulocytes inischemia-reperfusion injury. Am J Physiol 1988;255(6 Pt2):H1269-75.

19. Mathews WR, Guido DM, Fisher MA, Jaeschke H. Lipidperoxidation as molecular mechanism of liver cell injury dur-ing reperfusion after ischemia. Free Radic Biol Med1994;16:763-70.

20. Luo XP, Yazdanpanah M, Bhooi N, Lehotay DC.Determination of aldehydes and other lipid peroxidationproducts in biological samples by gas chromatography-massspectrometry. Anal Biochem 1995;228:294-8.

21. Montgomery JA, Jette M, Huot S, Des RC. Acyloin produc-tion from aldehydes in the perfused rat heart: the potentialrole of pyruvate dehydrogenase. Biochem J 1993;294(Pt3):727-33.

22. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF,Roberts LJ 2nd. A series of prostaglandin F2-like compoundsare produced in vivo in humans by a non-cyclooxygenase,free radical-catalyzed mechanism. Proc Natl Acad Sci U S A1990;87:9383-7.

23. Morrow JD, Roberts LJ 2nd. The isoprostanes. Currentknowledge and directions for future research. BiochemPharmacol 1996;51:1-9.

24. Morrow JD, Frei B, Longmire AW, Gaziano JM, Lynch SM,Shyr Y, et al. Increase in circulating products of lipid peroxi-dation (F2-isoprostanes) in smokers. Smoking as a cause ofoxidative damage. N Engl J Med 1995;332:1198-203.

25. Roberts LJ 2nd, Morrow JD. The isoprostanes: novel mark-ers of lipid peroxidation and potential mediators of oxidantinjury. Adv Prostaglandin Thromboxane Leukotriene Res1995;23:219-24.

26. Lindsay TF, Memari N, Ghanekar A, Walker P, Romaschin A.Rupture of an abdominal aortic aneurysm causes priming ofphagocytic oxidative burst. J Vasc Surg 1997;25:599-610.

27. Roberts LJ 2nd, Morrow JD. The generation and actions ofisoprostanes. Biochim Biophys Acta 1997;1345:121-35.

28. Lindsay T, Walker PM, Mickle DA, Romaschin AD.Measurement of hydroxy-conjugated dienes after ischemia-reperfusion in canine skeletal muscle. Am J Physiol 1988;254(3 Pt 2):H578-83.

29. Romaschin AD, Rebeyka I, Wilson GJ, Mickle DA.Conjugated dienes in ischemic and reperfused myocardium:an in vivo chemical signature of oxygen free radical mediatedinjury. J Mol Cell Cardiol 1987;19:289-302.

30. Morrow JD, Roberts LJ 2nd. Quantification of noncy-clooxygenase derived prostanoids as a marker of oxidativestress. Free Radic Biol Med 1991;10:195-200.

31. Catella F, Reilly MP, Delanty N, Lawson JA, Moran N,Meagher E, et al. Physiological formation of 8-epi-PGF2alpha in vivo is not affected by cyclooxygenase inhibition.

JOURNAL OF VASCULAR SURGERYVolume 30, Number 2 Lindsay et al 227

Adv Prostaglandin Thromboxane Leukotriene Res 1995;23:233-6.

32. Morrow JD, Awad JA, Kato T, Takahashi K, Badr KF,Roberts LJ 2nd, et al. Formation of novel non-cyclooxyge-nase-derived prostanoids (F2-isoprostanes) in carbon tetra-chloride hepatotoxicity. An animal model of lipid peroxida-tion. J Clin Invest 1992;90:2502-7.

33. Reilly MP, Delanty N, Roy L, Rokach J, Callaghan PO,Crean P, et al. Increased formation of the isoprostanesIPF2alpha-I and 8-epi-prostaglandin F2alpha in acute coro-nary angioplasty: evidence for oxidant stress during coronaryreperfusion in humans. Circulation 1997;96:3314-20.

34. Delanty N, Reilly MP, Pratico D, Lawson JA, McCarthy JF,Wood AE, et al. 8-epi PGF2 alpha generation during coro-nary reperfusion. A potential quantitative marker of oxidantstress in vivo. Circulation 1997;95:2492-9.

35. Thiagarajan RR, Winn RK, Harlan JM. The role of leukocyteand endothelial adhesion molecules in ischemia-reperfusioninjury. Thromb Haemost 1997;78:310-4.

36. Banda MA, Granger DN. Mechanism and protection fromischemic intestinal injury. Transplant Proc 1996;28:2595-7.

37. Johnston KW. Multicenter prospective study of nonrupturedabdominal aortic aneurysm. Part II: variables predicting mor-bidity and mortality. J Vasc Surg 1989;9:437-47.

38. Vedder NB, Fouty BW, Winn RK, Harlan JM, Rice CL. Roleof neutrophils in generalized reperfusion injury associatedwith resuscitation from shock. Surgery 1989;106:509-16.

39. Barroso-Aranda J, Schmid-Schonbein GW, Zweifach BW,Engler RL. Granulocytes and no-reflow phenomenon in irre-versible hemorrhagic shock. Circ Res 1988;63:437-47.

40. Welbourn R, Goldman G, O’Riordain M, Lindsay TF,Paterson IS, Kobzik L, et al. Role for tumor necrosis factor asmediator of lung injury following lower torso ischemia. JAppl Physiol 1991;70:2645-9.

Submitted Dec 7, 1998; accepted Mar 19, 1999.

JOURNAL OF VASCULAR SURGERY228 Lindsay et al August 1999

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