Differences between arterial and mixed venous levels of plasma hydroperoxides following major thoracic and abdominal operations

Free Radical Biology & Medicine, Vol. 9, pp. 485-494, 1990 Printed in the USA. All rights reserved.

0891-5849/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc

-~" Original Contribution DIFFERENCES BETWEEN ARTERIAL AND MIXED VENOUS LEVELS OF PLASMA HYDROPEROXIDES FOLLOWING MAJOR THORACIC AND ABDOMINAL OPERATIONS

RICHARD R . KEEN,* LISA A . STELLA,t D . PRESTON FLANIGAN,* a n d WILLIAM E . M . LANDSt *Departments of Surgery and tBiological Chemistry, University of Illinois College of Medicine at Chicago, West Side Veterans Administration Hosptial and Cook County Hospital, Chicago, IL 60612, USA (Received 10 May 1990; Revised and Accepted 21 August 1990) A b s t r a c t - - F a t t y acid hydroperoxides in the plasma of 18 patients who were undergoing normal postoperative periods following major thoracic or abdominal operations were measured by using a sensitive assay based upon the activation of the cyclooxygenase activity of prostaglandin H synthase. Following major thoracic operations of nine patients, the mean difference between the arterial (0.49 - 0.13 ixM, mean _+ S.E.M.) and mixed venous ( - 0 . 0 9 -+ 0.12 ixM) level of hydroperoxide was 0.58 _+ 0.13 IxM (p < 0.01). In marked contrast to this result, major abdominal operations of nine patients led to a mean difference between the arterial ( - 0 . 1 9 -+ 0.16 ~M) and mixed venous (0.46 -+ 0.08 p~M) hydroperoxide levels of - 0 . 6 5 _+ 0.17 IxM (17 < 0.01). Both pulmonary and intraabdominal tissues appear capable of generating significant amounts of fatty acid hydroperoxide in response to standard surgical procedures. The A-MV differences suggest that the blood-borne hydroperoxides were rapidly cleared from the circulation by tissue capillary beds.

Keywords--Free radicals

INTRODUCTION

peroxide in inflammatory cell systems can react with unsaturated lipids in the presence of iron salts to form lipid hydroperoxide in amounts sufficient to enhance prostaglandin synthesis. 4 Both hydrogen peroxide and fatty acid hydroperoxides appear able to facilitate the reaction of arachidonate with cyclooxygenase~l't2 and lipoxygenase, t3 The initial reaction products of both cyclooxygenase (prostaglandin G2) and 5-1ipoxygenase (5hydroperoxyeicosatetraenoic acid; 5-HPETE) are hydroperoxides that can stimulate even faster intracellulax oxygenation reactions, 14 forming more hydroperoxide and active eicosanoids. Thus, the local concentration of hydroperoxides (sometimes called peroxide tone tS) might be an important factor influencing the speed of eicosanoid formation in response to leukocyte activation at inflammatory sites. Thoracotomies and celiotomies are two categories of major surgical procedures that can create regional pulmonary and abdominal tissue injury. There is currently no clear knowledge of the degree to which lipid hydroperoxides may be produced by local inflammatory responses during the process of injury and healing. While investigating plasma hydroperoxides in the early postoperative period, we found elevations of hydroperoxides in plasma from the regions following the operations.

Many different effector molecules participate in inflammatory events, and understanding the network of eicosanoid and cytokine signals is essential to the interpretion of complex disorders such as multiple organ failure t or adult respiratory distress syndrome2 and to the design of effective new drugs. 3 The successful application of nonsteroidal anti-inflammatory drugs to many situations has led us to consider more closely the ways in which prostaglandins, and the hydroperoxides that activate prostaglandin biosynthesis, participate in important tissue responses. 2'4 We have regarded the generation of active oxygen metabolites (especially H202) as a pivotal process. 4 Both activated neutrophils and macrophages produce large amounts of hydrogen peroxide s'6 and superoxide 7'8 after exposure to a variety of stimuli, and hydrogen peroxide can directly augment cell prostaglandin production. 9"~° In addition, hydrogen peroxide and suPlease address correspondence to Richard R. Keen, Department of Surgery, (M/C 957), The University of Illinois at Chicago, 1740 W. Taylor St. Suite 2200, Chicago, IL 60612. Sources of financial support are a USPHS grant (HL-34422) and a Pfizer Biomedical Research Award (WEML) 485

R.R. KEENet al.

486 METHODS

Patient selection and sample collection Eighteen patients who had undergone major thoracic or abdominal operations in the previous 5 days and had radial artery catheters and pulmonary artery catheters placed for perioperative hemodynamic monitoring (SwanGanz Thermodilution Catheter, American Edwards Laboratories, Anasco PR) were studied. The nine thoracic surgical patients were in their first or second postoperative day, whereas the patients who had undergone abdominal procedures were in their first to fifth postoperative day. None of the patients in this study exhibited any signs or symptoms of sepsis on the day blood was drawn, and none subsequently developed positive bacterial blood cultures. Five mL of arterial blood from the radial artery and five mL of mixed venous (abdominal and peripheral venous) blood from the pulmonary artery was drawn, added to citrated Vacutainer tubes (Becton Dickinson Systems, Rutherford, NJ), and transported on ice. There was an average transport time of 10 min. Samples were centrifuged at 1500 g for 30 rain at 4°C, and the plasma was transferred to a new tube and kept on ice until further processing for the assay. Fresh peripheral venous blood obtained from normal volunteers was used in experiments to measure the levels of fatty acid hydroperoxide in peripheral venous plasma and to determine the stability of fatty acid hydroperoxide in plasma over time. In addition, preparations of pooled plasma from several volunteers were divided into 4-mL portions and stored at - 4 0 ° C for subsequent use as a matrix in the preparation of standard curves.

Preparation of working solutions Purified prostaglandin H (PGH) synthase was prepared from sheep seminal vesicles as described earlier. 16 The PGH synthase solution (15.5 units/~L; 0.12 mg/mL) for assays was prepared from the stock PGH synthase (30 IxM; 300 units/p~L) by dilution with buffer (20mM phosphate buffer, pH = 7.2 in 30% glycerol) so that 9.0 p~L of PGH synthase solution contained about 140 units of enzyme activity. PGH synthase units are defined as nmol 02 consumed/min. Arachidonic acid in toluene (50 p~L portion of 300 mM stock) (Nu-Chek Prep., Elysian, MN) was dried under a nitrogen stream, dissolved in 60 txL of 100% ethanol, and resuspended in 14.6 mL of 0.1 M Tris chloride buffer, pH 8.5. The aqueous arachidonate solution (lmM) was rendered peroxide-free by treatment with glutathione peroxidase (100 IxL of 0.2 units/p~L) and 150 ILL of 100mM reduced glutathione (GSH) (Sigma Chemical, St. Louis, MO) at 25°C for 5 rain and then stored on ice in a foil-covered tube. The assay mixtures described below contained sufficient NEM to

prevent interference by any GSH carried over. Aqueous solutions of 80 mM N-ethylmaleimide (NEM) (Sigma) and 40 mM GSH were made the day of the experiment, and a portion was combined to make a solution of 1:1 (v/v) 40 mM GSH:80 mM NEM. This solution was allowed to sit at 25°C for 30 min prior to use in plasma or buffer treatments. A standard hydroperoxide, 15-hydroperoxy- eicosatetraenoic acid (15-HPETE) was prepared by the method of Graft 17 and stored as a 0.07 mM solution in 100% ethanol at - 2 0 ° C . The concentration of the 15-HPETE was determined by its absorbance at 237 nm using a molar absorbance coefficient of 0.028/IxM. The purity was assessed by the symmetry of its absorbance spectrum between 222 and 252 nm.

Experimental design to measure plasma hydroperoxide Although the sequential order by which GSH, NEM, glutathione peroxidase, and ethanol were added to plasma or buffer differed for different assay strategies, the final amounts of these added materials were 2 p~mol GSH, 4 txmol NEM, 4 units glutathione peroxidase, and 30 p~L ethanol per mL of treated plasma or buffer. Total plasma cyclooxygenase activator ~8 was determined with 0.5 mL of each plasma sample that was treated with 50 p~L of the 1:1 (v/v) mixture of 40 mM GSH:80 mM NEM vortexed together and incubated for 15 rain at 25°C. Then 10 ixL of glutathione peroxidase (0.2 units/~L) and 15 ixL of ethanol were added to the treated samples, which were then vortexed, treated with two volumes (1 mL) of cold ethanol, and centrifuged at 600 g for 10 min at 4°C. The plasma supernatants ( - 1 . 2 mL) were transferred to separate tubes and placed on ice until they were used in assays for the determination of total cyclooxygenase activator later that day. Peroxidase-resistant cyclooxygenase activator, 19 was determined following treatment with 25 p~L of 40 mM GSH and 10 p~L of glutathione peroxidase (0.2 units/ ixL) added to 0.5 mL of each plasma sample and incubated for 15 minutes at 25°C. Then 25 IxL of 80 mM NEM and 15 IxL of ethanol were added to the plasma samples, which were vortexed with two volumes (1 mL) of ice cold ethanol, and centrifuged at 600 g for 10 rain at 4°C. The plasma supernatants ( - 1 . 2 mL) were removed to separate tubes and placed on ice until they were used for the determination of peroxidase-resistant activator later that day. Assaying the supernatants obtained from samples processed by these two methods provided a measure of the total cyclooxygenase activator and the peroxidaseresistant activator. The difference between these two values is the glutathione peroxidase-sensitive, or reducible, activator. This reducible activator most likely represents fatty acid hydroperoxides. Supernatants obtained from a similar treatment of phosphate buffer provided

Hydroperoxidesin postoperativeplasma plasma-free controls. 1 mL of 0.1 M phosphate buffer, pH = 7.2, was treated with 100 ixL of the 1:1 (v/v) mixture of 40 mM GSH:80 mM NEM and incubated for 15 min at 25°C. Glutathione peroxidase (20 IxL of stock; 0.2 units p~L) and 30 p,L of ethanol were added to the buffer, vortexed, treated with two volumes (2 mL) of cold ethanol, and centrifuged at 600 g for 10 min at 4°C. The supematant ( - 2 . 4 mL) was transferred to a separate tube, placed on ice, and used as a negative control for assays throughout that day. A second 1-mL sample of phosphate buffer was processed in an identical manner except that 30 txL of 0.07 mM 15-HPETE (in ethanol) was substituted for the 30 txL of ethanol added to the negative control sample, forming a solution of 2 I~M lipid hydroperoxide in buffer. The buffer containing the 15-HPETE was treated with two volumes ( 2 mL) of cold ethanol and centrifuged at 600 g for 10 rain at 4°C. The supematant was transferred to a separate tube, placed on ice, and used as a positive control for assays that day. Aliquots of the plasma superuatants were assayed for their ability to stimulate cyclooxygenase using an oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH). Each supernatant to be assayed (300 IxL) was added to a 3.00 mL reaction mixture formed from 2.030 mL of 1 mM NEM in 0.1 M potassium phosphate buffer (pH = 7.2), 300 p~L of 10 mM phenol in 0.1 M phosphate buffer, 30 IxL of 1 M NaCN, 40 p,L of 100 mM NEM, and 300 IxL of 1 mM arachidonate solution. Final concentrations of reagents in the cuvette were 1 mM phenol, 10 mM sodium cyanide, 2 mM NEM, 100 IxM arachidonate, and 6.7% ethanol. The solution in each cuvette was equilibrated for at least 2 rain at 25°C with the magnetic stirrer maintained at a constant speed before the 9.0 p,L of PGH synthase ( - 1 4 0 units) was added. Complete and reproducible transfer of the enzyme is needed because the rate and extent of oxygen consumption is directly related to the total amount of enzyme added. To achieve this, 1.0 IxL of a 30% glycerol in water solution was drawn into the syringe first to insure that the entire 9.0 IxL of enzyme solution was transferred to the cuvette. Between each incubation, the syringe was rinsed with 30% glycerol in water to avoid any carryover of enzyme, and the stock enzyme solution was mixed to ensure uniformity. The lag time (the time required for the enzyme to reach optimum velocity) was determined for each sample from the chart recording of oxygen consumption versus time as described elsewhere (Miller, J.F., Kulmacz, R.J., Lands, W.E.M. 1990. Timer for Hydroperoxide Assays. Anal. Biochem., Submitted). The positive buffer controls with added hydroperoxide were assayed at the beginning of each day to ensure that the system was responding adequately. The negative buffer control was assayed approximately six times throughout the day.

487

Supernatants of plasma obtained from patients were assayed between two and four times. The final values for lag times represented the average of these runs. Each 300 IxL of plasma superuatant contained 194 p,L of ethanol, and 11 IxL of aqueous additives (GSH, NEM, and glutathione peroxidase). Thus, the total picomoles of activator calculated for each assay represented the amount in an initial plasma volume of 95 p~L. Buffer supernatants (negative controls) were used routinely as an arbitrary reference point because of their simplicity. The difference in lag times between the buffer supernatant and the supernatant obtained from plasma initially treated with 1:1 GSH:NEM represented a decrease in lag time that can be attributed to the amount of total cyclooxygenase activator relative to that in the buffer system. The difference between the lag time of the buffer supernatant and that of the plasma initially treated with glutathione peroxidase in the presence of GSH represented material designated as peroxidase-resistant activator relative to the buffer system. The difference in lag times between the sample initially treated with glutathione peroxidase (resistant activator) and that initially treated with GSH plus NEM (total activator) represented the contribution of the peroxidase-sensitive, or reducible, cyclooxygenase activator, which is attributable to fatty acid hydroperoxide. This difference was expected to be zero when no glutathione peroxidasesensitive activator (reducible activator) was present in a sample. Statistical analysis was performed using Student's t test for paired samples.

RESULTS

Enzymatic assay for hydroperoxides After the addition of PGH synthase to a reaction cuvette, there was a short period of little detectable oxygen consumption that was followed by an accelerating phase in which the enzyme velocity became optimal and a decelerating phase in which the oxygen consumption became optimal (Fig. 1). The acceleration of the oxygenase activity has been attributed to stimulation by the accumulating 15-hydroperoxy endoperoxide, PGG z. 14 After the optimal velocity was reached, oxygen consumption slowed as the reaction-induced inactivation of the enzyme lz occurred. Sodium cyanide (10 mM) inhibited the enzyme and increased the lag time. 18 When picomole amounts of fatty acid hydroperoxide were initially added to the reaction cuvette, the inhibition of the enzyme was decreased, as determined by a measurably shorter lag time. The cyanide controls (zero picomoles of added hydroperoxide) consistently gave lag times between 100 and 160 s, although in each day's experiments, these control lag times differed slightly depending on the concentration of the PGH synthase used that day.

488

R.R. KEEN et al. (IIa) o

E

lag t i m e ( s ) - 129s - 0.145(s/picomoles HPETE)

E3 LO

m O'3 Z 0

20-

(IIb)

5O

0

°1o

× 0 © 0

30

60 90 120 TIME (sec)

150

Fig. 1. Enzymatic assay for hydroperoxides. Following the addition of PGH synthase, the oxygen electrode detected a short period of little oxygen consumption (0-30 sec) that was followed by a phase of maximal oxygen consumption (30-90 sec) and then decreased oxygen consumption ( > 9 0 sec). Cyclooxygenase that was inhibited by sodium cyanide (10 ixM) was stimulated by 50 and 100 picomoles of hydroperoxide.

Recovery of added hydroperoxide Standard curves were prepared in buffer, with and without glutathione peroxidase treatment, and in pooled plasma with and without glutathione peroxidase treatment. The cumulative buffer standard curve was prepared from 152 assays performed on buffer that contained exogenous 15-HPETE in concentrations of 0 to 2 IxM, and 86 control assays were done on buffer containing the same concentrations of 15-HPETE which had been treated with glutathione peroxidase (4 units/mL) following addition of the 15-HPETE. Similarly, recoveries were estimated from 152 assays with 15-HPETE (0 to 2 p~M) added to pooled plasma, and 80 control assays were performed on the pooled plasma containing similar concentrations of 15-HPETE which had been treated with glutathione peroxidase (4 units/mL). As the amount of 15-HPETE added to assay mixtures increased from 0 to 200 picomoles, the lag time decreased in a linear manner, whereas the decrease with peroxidase-treated samples was essentially zero, indicating that the added activator had been removed by treatment with the peroxidase. Regression analysis of combined data for the decrease in lag time with increased concentration of 15-HPETE in 152 assays with a buffer matrix and 86 assays with peroxidase-treated systems fitted the formulas: picomoles activator in 95 I-tL buffer = lag time(s) - 149 s - 0.245(s/picomoles HPETE) (Ib)

r= -0.751

treated picomoles of activator in 95 tzL buffer = lag time(s) - 140 s 0.023(s/picomoles HPETE)

r=0.102

r = - 0.696

treated picomoles of activator in 95 IxL plasma = lag tims(s) - 125 s - 0.017(s/picomoles HPETE)

z to (..9 ).-

(Ia)

picomoles activator in 95 FL plasma =

r = - 0.106

To validate the analytical procedures each day, the standard curve for positive buffer controls each day was compared with the composite buffer standard curve. The slope of the daily buffer standard curve was within the range of variability ( - 0 . 2 4 5 _+ 0.071 s/pmol 15HPETE) on each day when patient plasma was assayed. The pooled plasma that was used to provide a reproducible protein-rich matrix consistently produced shorter lag times (129 s) than the buffer (149 s) for the samples with no added activator and for samples treated with peroxidase. Apparently, it contained a cyclooxygenase activator that was resistant to glutathione peroxidase. The lower slope ( - 0 . 1 4 5 ) for the response to HPETE added to plasma indicated that the protein-containing matrix lowered the sensitivity of the assay so that a decrease of 10 s in lag time required about 70 picomoles compared to only 40 picomoles in the buffer matrix. The similar ordinate intercepts for the untreated (129 s) and treated (125 s) standard curves in pooled plasma (peripheral venous) indicated that the stored, pooled plasma did not contain sufficient peroxidase-sensitive activator to be detected. This lack of hydroperoxide also had been observed previously for samples that had been frozen and thawed several times, although the peroxidase-resistant activator in such samples had not been studied sufficiently before this. During other experiments, the pooled plasma used to make standard curves was also assayed on 20 additional separate occasions, confirming the negligible calculated value for fatty acid hydroperoxide ( - 0 . 0 8 -+ 0.13 txM). Fresh peripheral (antecubital) venous plasma obtained from apparently healthy volunteers on five separate occasions gave similar mean values for total activator (0.67 ± 0.24 txM) and peroxidase-resistant activator (0.74 ___ 0.21 p~M). Thus, these samples also contained no significant hydroperoxide ( - 0 . 0 7 --- 0.05 I~M). We noted that the average calculated amount of peroxidaseresistant activator (0.74 bM) in these samples was actually less than in the samples exposed to the freezethaw process (1.4 txM), although the calculated amount ofhydroperoxide was still essentially zero ( - 0.07 -+ 0.05 and - 0 . 0 8 ± 0.13). To determine whether or not a fatty acid hydroperoxide in plasma would degrade in a time-dependent manner, 15-HPETE was added to fresh peripheral venous

Hydroperoxides in postoperative plasma

489

Table 1. Cyclooxygenase Activator~ Concentration (ixM) Following Thoracic Operations

Patient (Age) T1 (24) T2 (65)

T3 (63) T4 ( 3 1 ) T5 (68) T6 (60) T7 (56) T8 (67) T9 (66) Mean _+ S.E.M.

Operation Thoracotomy for trauma CABG b

Arterial (A)

Mixed Venous (MV)

Reducible Activator

Reducible Activator

A-MV Difference Total Activator

Reducible Activator

2.48-1.93)

0.55

(2.00-2.00)

0.00

0.48

0.55

0.90-0.55)

0.35 1.38 0.41 0.00 0.42 0.83 0.34 0.14

(0.62-0.41) (0.76-0.76) (0.90-1.59) (0.21-0.69) (0.48-0.55) (0.97-0.48) (0.69--0.83) (0.83-0.97)

0.21 0.00 -0.69 -0.48 -0.07 0.49 -0.14 -0.14

0.28 1.03 -0.14 0.27 0.49 0.00 0.55 0.41

0.14 1.38 1.10 0.48 0.49 0.34 0.48 0.28

- 0 . 0 9 _+ 0.12

0.37 -+ 0.11 c

0.58 + 0.13 c

CABG 1.79-0.41) Thoracotomy L76--0.35) CABG (0.48-0.48) CABG (0.97-0.55) CABG (0.97-0.14) CABG (1.24-0.90) CABG (1.24-1.10)

0.49 -+ 0.13

aAll assay values represent the mean of 2 to 4 determinations. Values in parentheses are (total- resistant activator) from which the reducible activator (hydroperoxide) was calculated. bCABG; coronary artery bypass graft. Cp<0.01 by paired t test.

plasma from an apparently normal volunteer to give an expected final concentration of 400 pmol 15-HPETE/ 100 txL plasma. Aliquots assayed at 0, 40, and 120 min while standing at 4°C demonstrated a time-dependent loss of hydroperoxide: amount = 347 pmol- (time) x (1.79 pmol/100 txL/min); r = 0.987. This result suggests that 70 pmol of fatty acid hydroperoxide per 100 p~L of plasma might be consumed during the 40 min customarily used to process each plasma sample. A confinning experiment was conducted by another researcher in our lab during the following year. Hydroperoxide (15-HPETE) was added to fresh plasma to give an expected concentration of 77 pmol 15-HPETE/100 IxL plasma. Aliquots assayed at 0, 10, 25, and 60 min while standing at 4°C gave the relationship: amount = 80.8 pmol- (time) x (1.77 pmol/100 IxL/min); r = 0.984. The similar rates of loss ( - 1.79 vs. - 1.77) indicated a rate of consumption of fatty acid hydroperoxide in the plasma matrix that was independent of hydroperoxide concentrations.

Plasma hydroperoxides following thoracic operations Eight of the nine patients listed in Table 1 were in their first postoperative day, and six had undergone coronary artery bypass grafting (CABG) the previous day. Of the two other patients studied on the first postoperative day, one (T4) had undergone surgical therapy for an aberrant cardiac conduction pathway, whereas the second (T1) had undergone a thoracotomy for multiple gunshot wounds. One patient (T8) was in his second postoperative day following CABG. The mean age was 56, and eight patients were male and one (T4) female. Following thoracic procedures (Table 1), the level of total activator in arterial plasma (1.20 ___ 0.20 p~M; mean ___ S.E.M.) ranged from 2.48 ixM to 0.48 p~M.

The level of arterial activator that was resistant to glutathione peroxidase was less: (0.71 + 0.18 ixM), ranging from 1.93 IxM to 0. t4 ixM. The level of reducible activator (fatty acid hydroperoxide) in the arterial plasma samples following thoracic surgery (0.49 _ 0.13 IxM) ranged from 0 to 1.38 t~M. Mixed venous samples from these patients had a lower level of total activator that ranged from 2.00 ixM to 0.21 ixM with a mean value of 0.83 __+ 0.17 txM. In contrast, the mean level of resistant activator, 0.92 ___ 0.18 txM, was similar to that in arterial samples. Thus, the difference between these values indicates a neglibible mean level of reducible activator (fatty acid hydroperoxide) in the mixed venous plasma samples following thoracic operations ( - 0 . 0 9 --_ 0.12 I~M). Hydroperoxides were detected in the arterial plasma of all thoracic surgical patients studied except one (T5), Conversely, hydroperoxides were detected in only two (T2 and T7) of the nine mixed venous samples from these patients. The mean A-MV (arterial minus mixed venous) difference between paired hydroperoxide levels was 0.58 __+ 0.13 ixM. Using the t test for correlated (paired) data, this difference is significant (p < 0.01).

Plasma hydroperoxides following abdominal operations The patients listed in Table 2 underwent a variety of both elective and emergency abdominal operations. No patient demonstrated signs of sepsis on the day hydroperoxides were measured, though one patient (A1) had been septic at the time of admission. Plasma samples were drawn primarily during the second postoperative day, though the range was from the first to fifth postoperative day. The mean age was 61, and eight patients were male and one (A2) female.

R.R. KEEYet al.

490

Table 2. Cyclooxygenase Activator Concentrations (ixM) in Plasma Following Abdominal Operations

Patient (Age)

Arterial (A) Reducible Activator

Operation

A1 (59) Cholecystectomy(3)a A2 (71) Distal Pancreatectomy(5) A3 (67) Aorto-bifemoral Bypass(3) A4 (71) Abdominal Aortic Aneurysm Resection(2) A5 (60) Pelvic Exenteration(l) A6 (27) Exploratory Laparotomy for Trauma(2) A7 (64) Subtotal Colectomy(1) A8 (63) Vagotomyand Pyloroplasty(2) A9 (63) Aorto-bifemoral Bypass(3)

Mixed Venous (MV) Reducible Activator

A-MV Difference Total Reducible Activator Activator

(0.41-0.62) (1.10-1.38)

0.21 -0.28

(0.55-0.07) (1.77-0.55)

0.48 0.62

-0.14 -0.07

0.69 -0.90

(1.17-1.17)

0.00

(1.59-1.10)

0.49

0.42

0.49

(0.83-1.59)

-0.76

(l.10-1.24)

-0.14

-0.27

-0.62

(0.41-1.17)

-0.76

(0.90-0.21)

0.69

-0.49

- 1.45

(0.90-0.90)

0.00

(1.17-0.83)

0.34

-0.27

-0.34

(0.76-0.00)

0.76

(0.76-0.28)

0.48

0.00

0.28

(1.174).48)

0.69

-0.96

- 1.17

(1.17-0.69)

0.48

0.27

-0.48

(0.21-0.69) (0.90-0.90)

Mean +_ S.E.M.

-0.48 0.00 0.19 +_ 0.16

0.46 + 0.08

-0.32 _+ 0 . 1 0

b

-0.65

+

0.17 b

aSamples were assayed on the postoperative day shown in parens. bp<0.01 by paired t test.

Following abdominal operations, the level of total activator detected in arterial plasma ranged from 1.17 IxM to 0.21 IxM with a mean of 0.74 ± 0.11 ixM; (mean ± S.E.M.). The glutathione peroxidase-resistant activator in arterial plasma had a mean concentration of 0.94 _ 0.16 ixM, and the paired differences in the arterial plasma samples of - 0 . 1 9 ± 0.16 txM suggested that no fatty acid hydroperoxide was present in arterial blood of these patients. Hydroperoxides were detected in only one (A7) of the arterial plasma samples, whereas they were detected in the mixed venous plasma of eight of the nine patients who had experienced abdominal operations. The mean concentration of total activator in mixed venous samples ranged from 1.59 IxM to 0.55 txM, with a mean of 1.06 _+ 0.10 ixM. However, the mean concentration of resistant activator of 0.60 + 0.13 ixM led to calculated hydroperoxide levels in the mixed venous plasma of patients following abdominal surgery of 0.46 -+ 0.08 txM. Overall, the mean difference between paired A - M V hydroperoxide levels following abdominal procedures was - 0 . 6 5 ± 0.17 IxM (p < 0.01) indicates the presence of greater amounts of hydroperoxide in the mixed venous plasma. DISCUSSION

Limitations o f hydroperoxide assays

The values that we calculated for fatty acid hydroperoxide ( - 0 . 0 7 + 0.05 IxM; mean + S . E . M . ) in pe-

ripheral (antecubital) venous plasma from apparently normal volunteers differs in two ways from that reported previously from this laboratory. 19 In this study, the peroxidase-resistant activator (0.74 + 0.20 p~M) was significantly higher than reported earlier, 19 making the calculated peroxidase-sensitive activator in peripheral venous samples correspondingly lower. The unexpected presence of significant amounts of peroxidase-resistant activator in the plasma samples in this study caused us to measure both total and resistant activator in all samples so that the content of fatty acid hydroperoxide (peroxidase-sensitive activator) was calculated for each sample. It was theoretically possible to consider only the total activator when evaluating differences in regional formation of hydroperoxides. Such a simplified approach permits less effort (one-half of the number of assays) to indicate a significant arterial-mixed venous difference for samples following thoracic operations (0.37 ± 0.11 ~M; mean ± S . E . M . , p < 0.01) that contrasts markedly from the significant negative difference for samples after abdominal operations ( - 0 . 3 2 _+ 0.10 txM; p < 0.01). However, the added analyses with glutathione peroxidase treatment provided a valuable assurance that there was indeed hydroperoxide (peroxidase-sensitive material) in the plasma samples coming from the regions perturbed by the surgical intervention. A variety of methods for measuring lipid hydroperoxides in biological fluids have been reported, but all of the techniques are limited by a low specificity, low sensitivity or both. 2° The thiobarbiturate assay for malon-

Hydroperoxidesin postoperativeplasma aldehyde is subject to interference, and applying the measurement of absorbance at 233 nm for conjugated dienes to assays with tissue and body fluids may include many UV-absorbing materials that are not lipid hydroperoxide.2~ We recently tested the applicability of a modified iodometric assay for quantitating hydroperoxides in human plasma (Cramer, G., J.F. Miller, J., R.B. Pendleton, W.E.M. Lands., 1989. Iodometric measurement of lipid hydroperoxides in human plasma. Anal. Biochem. Submitted.) and found significant amounts of oxidant that was resistant to removal by glutathione peroxidase and thus apparently was not fatty acid hydroperoxide. The iodometric method also did not seem useful for routine assays because it was not very sensitive, requiring a nanomole equivalent to respond significantly above the noise level. We found that detecting hydroperoxides in biological fluids by the cyclooxygenase activation assay was not as sensitive or selective with plasma samples as it was for pure enzyme reaction systems, requiring a minimum amount of 70 picomoleequivalents in plasma to be above the high noise level. Nevertheless, we find this method to be sufficiently sensitive to be useful, although it is a tedious method. The current finding of a negligible amount of peroxidase-sensitive hydroperoxide for either fresh or stored peripheral venous plasma agrees with a recent report (using an isoluminol chemiluminescence assay) which concluded that lipid hydroperoxide was not greater than 0.03 IxM in normal plasma. 22 Our experience leads us to emphasize that all of the analytical methods can become much more specific if they are compared with an appropriate paired sample from an alternate region and also compared with peroxidase-treated controls. Nevertheless, there is at this time no single valid test for hydroperoxides in biological samples that is rapid, sensitive, and convenient. Thus, our experience reaffirms the need for caution in interpreting results of all hydroperoxide assays, while at the same time we note the importance of vigorously pursuing the valuable clues provided by such assays. Only a limited number of patients have Swan-Ganz catheters for temporarily monitoring their cardiovascular status, and we had initially collected the samples described in this report to serve as further controls in an ongoing study of plasma hydroperoxide levels. However, the consistent pattern of differences between the arterial and mixed venous levels indicated that some previously unrecognized hydroperoxide-related event occurred following surgical procedures.

Comparison of tissue injuries The detection of elevated hydroperoxides in blooddraining postoperative thoracic and abdominal cavities suggests that both regions are capable of generating sig-

491

Fig. 2. Amplificationof intracellular and extracellularhydroperoxides. nificant amounts of hydroperoxide. In addition, the relative lack of measurable hydroperoxide in blood-draining noninjured regions indicates that the hydroperoxide may be rapidly cleared or metabolized from plasma by systemic and pulmonary capillary systems. The probable clearance by capillary systems suggests that we should not expect to detect fatty acid hydroperoxide in peripheral venous plasma obtained from the antecubital vein that drains only the hand and distal ann region. This conclusion places important constraints on the type of samples that may be regarded as useful for monitoring hydroperoxides in plasma. Our previous studies 4 on the generation of hydroperoxides in inflammatory conditions prompt us to emphasize that extracellular hydroperoxides (like those in plasma) can arise from reactions of the H202 that is released from activated leukocytes, neutrophils, and macrophages (see Fig. 2). Each of the two types of operations described in the present report probably involved events that caused distinct local increases in activated leukocytes in pulmonary or hepatic tissue. For example, a difference in neutrophil counts in the right and left atria with the reestablishment of the cardiopulmonary circulation after coronary artery bypass suggested a sequestration of neutrophils in the lungs. 23 Apparently, a complement-induced pulmonary injury could develop with damage to pulmonary endothelial cells by the activated leukocytes. 24 Thus, elevation of C3a and C5a can be an initiating step leading to a neutrophil-mediated pulmonary inflammation that is reflected by increased arterial hydroperoxides compared to mixed venous levels following the thoracic operations. The one thoracic patient (T1) who did not undergo cardiopulmonary bypass had sustained an extensive pulmonary contusion with direct injury of lung tissue as a result of a thoracic gunshot wound. Presumably the direct injury to lung tissue might have provided a local level of inflammation at least comparable to that occurring after extracorporeal circulation. Only one patient with abdominal surgery (A7) had relatively high levels of fatty acid hydroperoxide in the arterial circulation. This patient had preoperative chronic obstructive pulmonary disease that required oxygen therapy, and at that time high levels of arterial hydroperox-

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ides (0.68 p~M) were found with no measurable mixed venous hydroperoxides. Following subtotal colectomy for colonic adenocarcinoma, his mixed venous hydroperoxides increased to 0.48 ~zM, and his arterial hydroperoxides remained elevated (0.76 ~zM). We cannot rule out that an underlying chronic inflammatory pulmonary condition of this patient resulted in the continued formation of arterial hydroperoxides. Transient increases in plasma conjugated dienes and malonaldehyde (MDA) have been reported in arterial (but not venous) plasma at 1 and 4 h following endotoxin-induced lung injury in sheep. 25 Injured pulmonary tissue was suggested as the source of the increased arterial dienes, and it was hypothesized that splanchnic beds could clear the conjugated dienes from the plasma. 25 Elevated dienes were noted in that study only in the first 4 h following endotoxin injury, whereas we measure elevated hydroperoxides during the second postoperative day (on the average). Thus, our data suggest that regional formation of hydroperoxide was prolonged after the surgical procedures in humans. Handling the intestines of experimental animals consistently resulted in translocation of bacteria to the mesenteric lymph nodes, 26 and a transient portal vein bacteremia might readily arise from such translocations of gut microorganisms. 27 This phenomenon would directly expose blood-borne bacterial polysaccharide (e.g., endotoxin28) to the large population of resident hepatic macrophages (Kupffer cells) which line the hepatic sinusoids to provide a vigorous respiratory burst with released oxygen-derived free radicals,29 and lead to elevated hydroperoxides in mixed venous plasma. Our findings of significant negative A-MV differences during the normal recovery period following abdominal surgery indicate that the regional release of oxidized fatty acids may be greater than expected previously. Extracellular and intracellular hydroperoxides Most cells appear to contain prostaglandin H synthase, and leukocytes, platelets, and pulmonary tissue also contain lipoxygenases, so that any of a number of cell types might generate fatty acid hydroperoxides. This generation may then cause the intracellular concentration of fatty acid hydroperoxide to increase from the nanomolar levels that activate cyclooxygenase3° to micromolar levels of hydroperoxide that can be formed by the oxygenase (see Fig. 2). However, the intracellular peroxidases 31 will limit the amount of oxygenase-derived hydroperoxide that could be accumulated or released by cells. In contrast to the intracellular formation, the primary mechanism by which extracellular hydroperoxides are formed appears to be nonenzymatic as extracellular hydroperoxides were only made in response to released hydrogen peroxide when in the presence of exogenous lipid and iron. 4

Extracellular hydroperoxides may enhance inflammatory responses by promoting leukocyte chemotaxis. 12HPETE elicited a maximal neutrophil chemotactic response at 12 ~M, and exceeded by over 50% the maximal neutrophil chemotactic response of its hydroxy acid, 12-HETE, and correlated with intracellular increases in guanosine-3', 5'-cyclic monophosphate (cGMP). 32 When incubated in the presence of C5a (but not in its absence) low levels of hydroperoxide (0.03 ~M 12HPETE, but not 12-HETE) significantly increased neutrophil release of [3-glucuronidase. 32 In a similar manner, 0.12 p~M 12-HPETE significantly increased neutrophil release of lysozyme (p < 0.05). The effective concentrations of 12-HPETE (0.03 to 0.12 ~zM) were within the range of hydroperoxide measured in the plasma of our postoperative patients. Thus, evidence exists that intracellular and extracellular hydroperoxides can affect a large number of cellular functions, including enzyme activation and inhibition, eicosanoid syntheses, 33 and neutrophil chemotaxis and granule release. Hydrogen peroxide, lipid hydroperoxide, and tissue responses The cumulative information on the cross-talk that can occur between macrophages, lymphocytes, neutrophils, endothelial cells, and platelets continually points to the importance of the adhesion of activated leukocytes to endothelial surfaces as the means by which cells achieve the intense positive feedback associated with local pathologic inflammation. Adhesion facilitates transcellular metabolism between adjacent cells in ways not possible with isolated single cells, and the synthesis made possible by transcellular metabolism could stimulate further adhesion (e.g., platelets can form LTC 4 with LTA 4 obtained from neutrophils, ~4 and LTC 4 promotes adhesion35). Eicosanoid-related transcellular metabolic events are known for H202, 9 prostaglandin H2, 36 and leukotriene A4, 34'3v and lipid hydroperoxides may be another intermediate in the intimate interactions of cells at inflammatory loci (see Fig. 2). The adjacent adherent cells can form unusually high local levels of hydrogen peroxide and lipid hydroperoxide, and this high peroxide tone seems likely to promote even more rapid rates of eicosanoid biosynthesis 4 and leukocyte adhesion and chemotaxis. Elevated hydroperoxides have not been reported previously although major abdominal surgery is recognized to be associated with extra-abdominal organ complications. For example, postoperative pulmonary complications were observed38 in 47 of 455 abdominal procedures (10.3%), but in only 2 of 330 nonabdominal (0.6%). Postoperative pulmonary complications are not restricted to patients with preexisting pulmonary disease, although the incidence in this group may be tWO39 tO three 38 times

Hydroperoxides in postoperative plasma higher than in patients without p u l m o n a r y disease. Such complications have consistently been more frequent following a b d o m i n a l than n o n a b d o m i n a l operations, 4° suggesting that processes triggered by the abdominal operations contribute to the subsequent p u l m o n a r y complications. Similarly, postoperative peritonitis appears to be a more lethal disease than primary intraperitoneal sepsis, 41 as 36 of 60 patients (60%) with postoperative intraperitoneal sepsis died, compared to 31 of 116 patients (27%) with primary intraperitoneal sepsis. Not only was the mortality rate in postoperative peritonitis more than twice that seen with primary peritonitis, but intraperitoneal infection was the cause of death in a significantly greater percentage (86% vs. 61%) of postoperative patients. 39 It seems desirable to develop methods to monitor in more detail those signals that reflect regional inflammatory responses so that appropriate early intervention can be achieved. Our study demonstrated that hydroperoxides are measurable in plasma at a considerable distance from their probable site of formation, and they m a y be useful as a marker of regional leukocyte activation. In these experiments, the hydroperoxide formation occurred during normal recovery periods of postoperative patients. Thus, we seemed to be monitoring a subclinical level of oxidant production that was significant. Whether greater degrees of hydroperoxide formation are associated with the complications of the adult respiratory distress and multiple organ failure syndromes remains to be determined. Further experiments can now focus upon the degree to which such extracellular hydroperoxides m a y mediate cell responses in tissues distant from their origin.

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