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Am J Physiol Regul Integr Comp Physiol 284: R550-R557, 2003. First published October 24, 2002; doi:10.1152/ajpregu.00285.2002
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Vol. 284, Issue 2, R550-R557, February 2003

Protein-free phospholipid emulsion treatment improved cardiopulmonary function and survival in porcine sepsis

Roy D. Goldfarb1, Thomas S. Parker2, Daniel M. Levine2, Dana Glock1, Imran Akhter1, Azzam Alkhudari1, Robert J. McCarthy1, Eric M. David2, Bruce R. Gordon2, Stuart D. Saal2, Albert L. Rubin2, Gordon M. Trenholme3, and Joseph E. Parrillo1

Sections of 1 Critical Care Medicine and 3 Infectious Disease, Rush Medical College, Chicago, Illinois 60612; and 2 The Rogosin Institute and Departments of Biochemistry and Surgery, The New York Hospital-Cornell Medical Center, New York, New York 10021


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lipoprotein phospholipid (PL) plays a major role in neutralization of endotoxin. This study tested the hypothesis that prophylactic administration of a PL-enriched emulsion (PRE), which augments PL content of serum lipoproteins and neutralizes endotoxin in vitro, would preserve cardiovascular function and improve survival in porcine septic peritonitis. A control group was compared with low-, mid-, and high-dose treatment groups that received PRE by primed continuous infusion for 48 h. A fibrin clot containing live Escherichia coli 0111.B4 was implanted intraperitoneally 30 min after the priming dose. Survival increased in a dose-dependent manner and was correlated with serum PL. Infused PL was associated with high-density lipoprotein in the low-dose group and all serum lipoproteins at higher doses. Treatment significantly lowered serum endotoxin and tumor necrosis factor (TNF)-alpha , preserved cardiac output and ejection fraction, and attenuated increases in systemic and pulmonary vascular resistances. This study demonstrated that augmentation of lipoprotein PL via administration of PRE improved survival and offered a novel therapeutic approach to sepsis.

serum endotoxin; tumor necrosis factor; cardiac contractility; cardiac output; lipoprotein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS RECOGNIZED that elevated serum cholesterol is a risk factor for overall mortality when it is very high; it is also a mortality risk factor when it is very low (see review, Ref. 18). The benefits of reduced cardiovascular morbidity and mortality by treatment of hypercholesterolemia with cholesterol-lowering drugs are well documented. Low lipid and lipoprotein concentrations are associated with a poor prognosis in several studies of elderly individuals (20, 28, 34). Hypocholesterolemia was associated with development of infectious disorders over a 15-yr period in the Kaiser Permanente study of 15,000 healthy men and women (10) and with reduced hospital survival (35) and poorer clinical outcomes in a surgical intensive care unit (6). These data suggest that circulating lipoprotein may interact with mediators of systemic inflammation or bacterial products.

In fact, there are substantial data indicating that serum lipoproteins may neutralize circulating bacterial toxins. High-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride-rich lipoproteins, very low density lipoprotein (VLDL), and chylomicron remnants have all been shown to bind and neutralize bacterial endotoxin in vitro (3, 8, 33). Bacterial endotoxins are a diverse family of LPS that are shed from the outer membrane of gram-negative bacteria. Serum LPS-binding protein (LBP) presents LPS to CD14 and TLR-4 on cells of the monocyte/macrophage lineage (24, 36), which then initiate release of inflammatory mediators, causing systemic inflammatory syndrome and septic shock (1). Lipoproteins function as alternate neutralizing acceptors of LPS bound to LBP (12, 32, 37). We have shown that LPS neutralization correlates with the amount of phospholipid (PL) in lipid or lipoprotein particles (23). HDL, a primary carrier of PL among lipoproteins, has significant endotoxin-neutralizing capabilities. Infusion of HDL protects against lethal consequences of LPS administration in mice (15) and blocks LPS-induced cytokine production in rabbits (9) and human volunteers (21, 22).

This study tested the hypothesis that prophylactic administration of a protein-free, PL-rich lipid emulsion, which enriches serum lipoproteins with PL and neutralizes endotoxin in vitro, would provide a survival benefit in a porcine model of septic peritonitis (5). Pigs were chosen for this study because their serum lipoprotein distribution and sensitivity of their cardiopulmonary responses to systemic sepsis are closer to humans than are those of rat, rabbit, or dog. Septic peritonitis was modeled by introducing a 90% lethal dose of Escherichia coli 0111.B4 encased in a fibrin clot into the abdomen. This allows live gram-negative bacteria to grow and multiply within a protected nidus of infection, colonize the peritoneal cavity, and enter the systemic circulation causing bacteremia, endotoxemia, cytokinemia, and cardiopulmonary changes typical of septic shock. In whole human blood, we demonstrated (23) a dose-dependent effect of PRE to retard endotoxin-stimulated tumor necrosis factor (TNF)-alpha expression. In this study, multiple PRE infusion regimens were tested to establish a similar dose-dependent effect in vivo as well as to gain insight into minimal effective dose levels.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pigs

Healthy, normal Yorkshire pigs weighing 25-35 kg were obtained from Oak Hills Farms, a certified US Department of Agriculture supplier of laboratory animals. This protocol was reviewed and approved by Rush-Presbyterian-St. Luke's Medical Center's Animal Use Committee. Rush's staff veterinarians carefully monitored these experiments for adherence to National Institutes of Health guidelines for use of animals.

Experimental Protocols

Instrumentation surgery. Each pig was preanesthetized with ketamine (100 mg/kg) and glycopyrolate (0.016 mg/kg), intubated, and ventilated with isoflurane (1-2%) in oxygen to provide surgical anesthesia. With the use of sterile surgical techniques, ultrasonic crystals (Triton Technology) to measure short-axis diameter and a pressure transducer (Konigsberg Instruments) to measure left ventricular pressure were implanted in the left ventricle. A flow probe (Transonic) was placed around the pulmonary artery to measure cardiac output, and catheters were implanted in the pulmonary artery and aorta. The chest was closed, spontaneous respiration restored, and each animal was transported to postsurgical recovery where it was observed until full recovery.

Treatment. After 5-7 days of postsurgical recovery, basal cardiovascular and blood chemistry data were obtained (defined as day 0). On the next day, each animal was returned to the lab and animals were treated prophylactically with saline (n = 5), placebo (n = 7), or PRE (defined as day 1). A priming dose was delivered over 15 min followed by continuous intravenous PRE for 48 h. Three treatment groups were used: low (n = 7), mid (n = 7), and high (n = 6); all received a priming dose of 100, 200, or 200 mg PL/kg, respectively, followed by continuous infusion at 25, 25, or 100 mg PL · kg-1 · h-1, respectively. PRE or vehicle was infused through the pulmonary artery catheter. Total infusate volume was 2 liters on day 1 and 1 liter on day 2. Animals were observed continually for up to 72 h. Data were collated hourly on day 1 and daily on days 2, 3, and 4. No restraint was necessary during the 48 h of PRE infusion because sepsis reduced physical activity.

Control treatments: saline/placebo. Both 0.9% saline (n = 7) and a placebo (n = 5) were used as control treatments. The placebo solution was prepared by diluting 20% Liposyn 1:64 in 2.6% (wt/vol) glycerol. Lipid content of the placebo solution provides enough opacity to make it visibly indistinguishable from emulsion, but not enough lipid to neutralize endotoxin in human whole blood or to protect mice from a lethal endotoxin challenge (data not shown). Placebo and saline control solutions were administered in volumes equal to volumes received by high-dose group.

Emulsion. Fresenius Kabi (Clayton, NC) manufactured PRE used in this study. Soy phosphotidylcholine (Phospholipon 90G, American Lecithin) and soy triglyceride (Fresenius Kabi) were mixed (92.5% PC:7.5% TG) and emulsified in 18 mM sodium cholate (New Zealand Pharma), containing 2.6% glycerol wt/vol (Fresenius Kabi) by repeated passes through an APV Gaulin homogenizer at 10,000 PSI. The resulting emulsion, PRE, was sterilized by repeated passages through a 0.22-µm membrane and stored in glass bottles. The final mixture contained 100 mg PL/ml in 18 mM sodium cholate and 2.6% glycerol. PRE contained no protein.

Emulsion bioassay. PRE used in this study was tested for bioactivity in a whole human blood TNF-induction system that we have described previously (23). Briefly, a two-way dose-response experiment was carried out by mixing PRE with whole human blood and HBSS to provide final concentrations of PRE PL of 0, 0.1, 0.3, 1.0, 3.0, and 10 mg/ml in 50% whole blood. E. coli 0111:B4 LPS (List Biologicals) was added to give final concentrations of 0, 0.01, 0.1, 0.3, 1, 3, 10, and 100 ng/ml. Thirty-six (6 × 6) 1-ml incubations were kept at 37°C for 4 h, centrifuged at 2,000 g for 10 min, and the cell-free supernatants were stored at -70°C for measurement of TNF (Amersham Biosciences ELISA RPN 2148).

Septic peritonitis. After recording of basal parameters and delivery of the priming dose, each pig was anesthetized with sodium thiopental, an ultra-short-acting barbiturate. A small (2-3 cm) midline incision was made, and a bacteria laden fibrin-thrombin clot (100-150 ml) was inserted at a dose range of 2-3 × 1010 colony forming units (cfu)/kg E. coli 0111.B4. This dose of bacteria was selected to produce a 90% lethal dose peritonitis model. This level of insult was selected after power analysis so that any potential therapeutic effect of PRE infusion would be observable using the smallest number of animals. Animals recovered from thiopental anesthesia within 10 min. Each animal was observed for up to 72 h or until spontaneous expiration (15). Pigs did not receive antibiotic therapy, but did receive 2 liters of normal saline during the first 24 h and 1 liter on each subsequent day.

Endpoints. The primary endpoint of this study was mortality. Each animal was observed for 72 h or until spontaneous expiration. An experiment was terminated if cardiac output fell below 1 l/min for 30 consecutive min or if either the principal investigator or monitoring veterinarian (blinded to treatment) judged that an animal was in distress with no hope of recovery. Mortality was recorded at 24 and 72 h. Previous experience (5) demonstrated that no animals expired after 72 h and thus can be considered long-term survivors. Secondary endpoints were effects of treatment on serum TNF-alpha levels and measures of cardiac dynamics and cardiovascular status.

Cardiodynamic data acquisition and analysis. Measured variables recorded were left ventricular pressure (LVP), pulmonary artery blood flow (PAQ), pulmonary and arterial blood pressures (PAP and AoP), and short-axis diameter (D). From these measured variables, the following were calculated using software developed in this laboratory (4, 5): dP/dt max and min, cardiac output (l/min by integration of PAQ signal), systemic and pulmonary vascular resistance, heart rate (HR), peak LVP, end systolic diameter (ESD), end-diastolic diameter (EDD), and %diameter shortening [%DSh = (EDD - ESD/EDD)]. Cardiodynamic data were obtained every 15 min after implantation of bacteria-laden fibrin clot continuing for 6 h each day. These data were collated and indexed by time. Time 0 was defined to be all basal values. Data obtained on the day of implant were indexed by hour, 1-6. Data obtained on following days were indexed in 24-h intervals (24, 48, and 72 h).

Samples and Measurements

Blood and serum. Samples (10 ml) were obtained before PRE infusion (basal), 15 min (after the priming dose), 1, 2, 4, 6, 24, and 48 h after clot implant, and 1, 2, 3, and 4 h postinfusion (49-53 h after clot implant.) Blood was drawn from a central arterial line, allowed to clot, and immediately spun in a refrigerated centrifuge. Aliquots of serum were shipped on dry ice to The Rogosin Institute Clinical Research Laboratory for measurement and stored at -70°C.

Lipids. All lipid measurements were carried out with commercial kits adapted to run on a Roche COBAS FARA II (Roche Diagnostic Systems, Indianapolis, IN). Total cholesterol and triglycerides were measured by enzymatic analysis as described previously (17). Cholic acid was measured using an enzymatic test kit (Sigma Diagnostics, St. Louis, MO). PL was measured using an enzymatic method based on measurement of choline content of phosphotidylcholine, sphingomyelin, and lysophosphatidylcholine (Wako Chemical, Dallas, TX); these PLs comprise ~95% of total serum PLs.

Lipoproteins. Lipoproteins: VLDL, LDL, and HDL were separated by size-exclusion chromatography. Serum (200 µl) was injected onto an AKTA FPLC apparatus (Amersham Pharmacia Biotech), fitted with two Superose 6 size exclusion columns in series, and eluted with phosphate-buffered physiological saline (pH 7.0) at 0.5 ml/min (16). Fractions were collected (750 µl) and analyzed for cholesterol, PL, and triglyceride as described above. Ex vivo lipoprotein remodeling studies were done by incubating serum (200 µl) with saline, emulsion (final PL = +440 mg/dl) and/or BODIPY (Molecular Probes) R595 LPS (List Biological Labs), prepared as described by Yu and Wright (38) (final LPS = 1 ng/ml) at 37°C for 30 min. Lipoproteins were separated by flotation from 1.24 g/ml NaBr in a Beckman TL100 rotor at 100,000 rpm for 3 h at 20°C. Native gradient gel electrophoresis was done on 4-30% Isophore gradient gels from IsoLab (Akron, OH) as described by Nichols et al. (19). BODIPY-LPS and protein (SYPRO Orange) were imaged in a Molecular Dynamics Fluorimager.

Endotoxin. Serum endotoxin was measured by a kinetic limulus amebocyte lysate method (CX99002, Coamatic Endotoxin, Associates of Cape Cod, Falmouth, MA) according to the manufacturer's instructions. A Molecular Devices Thermomax microplate reader and SoftMax Pro software, version 2.6.1, were used to collect kinetic data (OD450/20 s for 20 min), estimate reaction lag times, and calculate unknowns. Samples were thawed, diluted 1:10 or more as needed, and heated to 75°C for 5 min to unmask bound endotoxin. Unknowns were analyzed with and without addition of purified E. coli 0111:B4 LPS (List Biologicals) as an internal standard. Recovery of an internal standard averaged 89 ± 23% and was not affected by PL concentration of serum samples. Serum endotoxin levels are reported in international endotoxin units as the mean of three or more measurements when the initial three measurements were within three standard deviations of their mean. Approximately 50% of samples failed this test. When this occurred, the measurement was repeated twice more and the mean was calculated from five measurements.

TNF-alpha . Pig TNF-alpha was measured using EP-TNF-alpha kits from Endogen (Woburn, MA) according to directions of the manufacturer.

Statistical analysis. Saline (n = 5) or placebo (n = 7) controls were combined into a single group after analysis of serum PL; endotoxin and TNF results indicated no between-group difference. Mortality was tested by chi 2 test. Survival times were analyzed by the Kaplan-Meier method with Wilcoxon chi 2 P values multiplied by three to correct for multiple comparisons of dose groups. Bacterial load, serum PL, endotoxin, and TNF-alpha were tested as covariates of survival by multivariate analysis by Cox proportional hazards method. Analysis of cardiodynamic data was affected by progressive reduction in animal numbers in all groups except the high-dose group. To minimize this affect, the control group (n = 12) was compared with all treated animals (n = 20) for analysis by two-way ANOVA with adjustment for number of surviving animals at each time point. Serum endotoxin comparisons are based on 2-h samples and TNF-alpha max was defined as the highest TNF-alpha measured in each animal in the interval between 30 and 120 min to avoid effect of dropout. Variables that were not normally distributed (e.g., serum LPS, TNF, and PC) were tested for differences by ANOVA on ranks. JMP version 4.0 (SAS, Cary, NC) was used for survival analysis; all other statistical testing was done with SigmaStat for Windows, version 2.03 by SPSS (Chicago, IL).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PRE contained 92.5% PL and 7.5% TG in a solution of 18 mM sodium cholate and 2.6% glycerol. In whole human blood, PRE (300 mg/dl as PL) increased concentration of LPS required to give 50% maximal TNF-alpha production by 10-fold (from <1.0 to >10 ng/ml). Stored PRE samples retained this level of bioactivity when retested after PRE batches were used in pigs. Preliminary studies in healthy pigs confirmed that PRE could be given to healthy pigs at doses up to 1,200 mg · kg-1 · 2 h-1 without significant toxicity.

Survival and Serum PL Concentrations

Emulsion was administered before (prophylactic) induction of septic peritonitis as a primed continuous intravenous infusion (CIVI) to maintain normal or supernormal serum PL concentrations during peritonitis. Serum PL levels in placebo (control) and PRE-treated animals are shown in Fig. 1, top. Serum PL in control pigs fell from basal [70 ± 25 mg/dl (n = 12)] to a low of 33 ± 1 mg/dl between 6 and 24 h postinfection. PRE treatment was successful in augmenting endogenous serum PL in all treatment groups. After priming and 4 h of CIVI, it was 164 ± 61 mg/dl in low-dose group (prime 100 mg/kg, CIVI 25 mg · kg-1 · h-1), 313 ± 120 mg/dl in mid-dose group (prime 200 mg/kg, CIVI 25 mg · kg-1 · h-1), and 585 ± 354 mg/dl in high-dose group (prime 200 mg/kg, CIVI 100 mg · kg-1 · h-1) compared with 67 ± 21 mg/dl in controls. Because both mid-dose and low-dose groups received PRE at 25 mg · kg-1 · h-1, their serum PL levels were similar between 4 and 48 h. The profile of serum PL concentrations was more complicated in the high-dose group and may reflect saturation of clearance between 8 and 24 h followed by a period of accelerated acute phase clearance. Serum PL concentrations returned to baseline within 24 h after halt of CIVI in all treated groups.


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  		Fig. 1.   Time course of serum phospholipid concentrations and survival. Top: serum phospholipid concentrations (mg/dl) are plotted as mean ± SE for each treatment group. , Controls; , low dose; black-triangle, mid dose; black-down-triangle , high dose. Arrows indicate the times of treatment events. Bottom: group survival rates (symbols as above) are shown as percent surviving. At the termination of the study, group survival rates were control 2/12 (16.7%); low dose 4/7 (57.1%, P = 0.051); mid dose 6/7 (87.5%, P = 0.04); and high dose 6/6 (100%, P = 0.001).

The Kaplan-Meier survival plot is shown in Fig. 1, bottom. All control and treatment groups received similar bacterial loads (overall mean 2.33 × 1010 ± 0.33 × 1010 cfu, between-group ANOVA: P = 0.711). This bacterial load produced a low survival rate in the combined control group. Only one of seven saline- and one of five placebo-treated pigs survived to 72 h for a total survival rate in the combined control groups of two of twelve or 16.7%. Survival rates in the treatment groups were low dose four of seven (57.1%, P = 0.051 vs. control), mid dose six of seven (87.5%, P = 0.04), and high dose six of six (100%, P < 0.001).

Serum TNF-alpha and Cardiopulmonary Responses to Sepsis and Treatment

Time courses of appearance of TNF-alpha in serum and cardiopulmonary indicators of the evolution of shock are shown in Fig. 2. Induction of septic peritonitis was followed by an increase in serum TNF-alpha concentrations in all animals that peaked between 1 and 1.5 h and returned to baseline by 3-4 h in surviving controls and all treated animals. Treatment lowered peak TNF-alpha concentrations by 68% from 2,251 ± 1,014 to 720 ± 355 pg/ml (P < 0.05). Cardiac output began to fall in control and treated animals shortly after the spike in serum TNF-alpha (Fig. 2B). The initial decline in cardiac output was preceded by sharp increases in systemic vascular resistance (Fig. 2C) and pulmonary vascular resistance (Fig. 2D) coincident with peak serum TNF-alpha levels. A continuing decline in cardiac output in controls is coincident with a second spike in pulmonary vascular resistance and a decline in ejection fraction (%diameter-shortening; Fig. 2E). Although pulse rate (Fig. 2F) increases in both groups, it is not sufficient to preserve cardiac output in the presence of relatively high vascular resistance that develops in untreated animals. Cardiac output recovered to basal or super-basal (hyperdynamic) levels on day 2 in surviving treated animals. These differences in cardiopulmonary function were clearly reflected by clinical signs of sepsis. Increased pulmonary vascular resistance was accompanied by development of pulmonary edema and noticeably labored breathing. Increased systemic vascular resistance and disseminated intravascular coagulation were evident by subcutaneous discoloration and hypothermia. Two survivors in the control group demonstrated these clinical signs for at least 48 h. Neither surviving control animals ate spontaneously; both had to be hand fed and watered. In contrast, treated animals rarely exhibited even mild subcutaneous discoloration or labored respiration. By 4 h nearly all treated animals had rosy pink skin tones characteristic of normo- or hyperperfusion. All surviving treated animals were drinking and eating spontaneously by 48 h, if not sooner.


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  		Fig. 2.   Tumor necrosis factor (TNF)-alpha and cardiopulmonary responses to sepsis. Serum TNF-alpha and cardiopulmonary changes in control (n = 12; ) and phospholipid-enriched emulsion (PRE)-treated (n = 20; ) groups are shown at times from baseline through 48 h. All results are reported as means ± SE. Data were tested for significance by 2-way ANOVA with the Bonferoni correction for multiple comparisons. Threshold for significance is P < 0.05. Time changes were significant in all groups. * Times with significant treatment effects. Vertical shaded area indicates the time of peak serum TNF-alpha . SVR, systemic vascular resistance; PVR, pulmonary vascular resistance; DSh, diameter shortening; heart rate is presented in beats/min (BPM).

Effect of Emulsion on HDL and Binding of Fluorescent Endotoxin

PRE PL was found to associate preferentially with serum to HDL (Fig. 3) at doses up to 200 mg · kg-1 · 2 h-1. Ex vivo mixing studies of emulsion and human serum demonstrated a size shift of HDL on native gradient gel electrophoresis (NGGE) consistent with binding of additional PL. Fluorescent BODIPY-LPS added to serum was bound to normal and PL enriched HDL after NGGE (Fig. 3).


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  		Fig. 3.   Binding of endotoxin to serum high-density lipoprotein (HDL) and phospholipid-enriched HDL. Fluorograms of HDL resolved by size on native gradient (4-30%) gel electrophoresis and stained for protein (green) or Re595 BODIPY LPS (red). Serum was incubated (30 min at 37°C) with and without PRE (dose equivalent to 200 mg/kg phospholipid) and/or BODIPY LPS [final (LPS) 1 ng/ml] and HDL separated by ultracentrifugation and native gradient gel electrophoresis as described in METHODS.

PL Distribution

Distribution of PL across lipoprotein fractions is shown for representative animals pre- and postsurgery and after 2 h of treatment in Fig. 4. Before surgery, most serum PL is in the HDL fraction in healthy animals. In the postsurgical acute phase, total serum PLs were reduced 30-40%, but the relative distribution of PL in VLDL, LDL, and HDL fractions was not changed. Treatment with placebo had no effect on distribution of PL between VLDL, LDL, and HDL compared with pretreatment or saline-treated controls. Infusion of emulsion preferentially increased HDL PL levels in the low-dose group. In the mid-dose group, peak broadening suggests increasing size heterogeneity in all lipoprotein fractions, and small increases in peak area for VLDL and LDL indicate more PL mass in these lipoprotein fractions. In the high-dose group, the capacity of serum lipoproteins to accept PL was clearly saturated: most PL eluted as large particles in VLDL size range. This large-particle fraction contained less triglyceride per unit PL compared with VLDL in the pretreatment samples, suggesting enrichment with PL vesicles or a lipoprotein-x-like particle (7, 14). The interpretation is consistent with the decreased PL peak area in the LDL and HDL fractions in the mid- and low-dose groups, which suggests that native lipoproteins and apolipoproteins had remodeled into the larger particles (29, 30).


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  		Fig. 4.   Distribution of phospholipid on serum lipoproteins. Data are FPLC size-exclusion chromatography profiles of serum lipoprotein phospholipid from animals representative of each control and dose group. All posttreatment samples are from the 2-h time point. Peaks are very low density lipoprotein (VLDL) and/or lipoprotein-x-like phospholipid vesicles 10-18 ml, low-density lipoprotein (LDL) 18- 26 ml, HDL 27-35 ml.

Serum Endotoxin

Serum endotoxin was measured during the 2nd h of septic peritonitis. Median endotoxin level in controls was 112 EU/ml (25% and 75% confidence limits: 4.22-500) compared with a median of only 3.0 EU/ml (25% and 75% confidence limits: 0.9-17.4) in the combined treatment groups (P < 0.001). Serum endotoxin levels tended to decrease with increased PRE dose, but large within-group variation of the serum endotoxin measurements and small numbers of animals within groups left the dose-group comparisons short of statistical significance (P < 0.07). Further analysis revealed that serum endotoxin concentrations were strongly related to serum PL concentration during the initial 2 h of treatment (see Fig. 5).


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  		Fig. 5.   Serum endotoxin and phospholipid. Data are serum endotoxin concentrations 2 h after induction of peritonitis plotted against serum phospholipid concentration at the same time. Circles, controls; squares, low dose; triangles, mid dose; inverted triangles, high dose. Filled symbols indicate animals that did not survive; open symbols indicate surviving animals (P = 0.03 for trend of endotoxin vs. phosphotidylcholine at 2 h).

Dose-Response Relationships

There was a strong dose-response relationship between serum PL and mortality (Fig. 6). Controls had the lowest PL concentration and the highest mortality. Serum PL concentrations increased across all treatment groups, and increased serum PL was associated with decreased mortality. For individual animals, survival was predicted best by serum PL concentration (at 0-4 h) and bacterial load (P < 0.001 and 0.049, respectively; Cox proportional hazard test). Neither serum endotoxin nor TNF-alpha max added significantly to the Cox proportional hazards model in stepwise multivariate analysis. Only 1 of 15 animals with serum PL concentrations >200 mg/dl died compared with 13 of the 17 animals with serum PL <200 mg/dl.


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  		Fig. 6.   Serum phospholipid concentrations and mortality. Data are mean and standard deviation of the serum phospholipid concentration through the first 4 h of treatment and percent mortality. Mortality rates (inverse of survival rates used elsewhere) were 10/12 (83%) in the control group, 3 of 7 (42.9%, P = 0.051 vs. control) in the low-dose group, 1 of 7 (12.5%, P = 0.04) in the mid-dose group, and 0 of 6 (0%, P < 0.001) in the high-dose group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrated the novel role of PL as a defense against bacterial toxins. These data confirm that serum lipoproteins participate in host defense. Mortality in this fatal model of septic peritonitis was inversely related to serum PL concentration. Elevation of serum PL was also associated with decreased serum TNF-alpha and preserved cardiopulmonary function. These data highlight the role of PL, but do not exclude a function for other serum proteins and lipoprotein components that have been shown to interact with LPS. PL was infused as an emulsion into the circulating blood volume, where it combined with LBP, HDL, LDL, and VLDL that were present in serum and formed remodeled particles. Our ex vivo mixing studies show that remodeled PL-enriched particles bind LPS.

This experiment employed a severe model of sepsis in which live gram-negative bacteria reproduced within a protected nidus of infection. From this protected peritoneal reservoir, bacteria and endotoxin enter the systemic circulation for at least 48 h, causing bacteremia, endotoxemia, cytokinemia, and cardiopulmonary changes typical of septic shock (5).

However, a number of features of clinical sepsis common in hospitalized populations could not be modeled. Clinical septic shock is often preceded by a period of several days of systemic inflammation and hyperdynamic sepsis. These animals were uniformly young and healthy with no other concurrent disease or trauma other than surgery 1 wk before induction of sepsis. In this model, the time of onset of infection was known and there were no prior or subsequent complications. It remains to be determined whether raising serum lipoprotein (PL) concentrations will be of benefit in the more complicated clinical setting.

Two observations illuminate the mechanisms of PRE's actions in this model. First, prophylactic administration of emulsion delivered PL preferentially to HDL in low- and mid-dose groups, yet optimal suppression of TNF-alpha and the survival benefit were most evident in the mid- and high-dose groups where much or most of the PL was associated with particles other than HDL. This suggests that the overall serum PL concentration is more important than the concentration of PL in any particular lipoprotein fraction.

The second new finding bearing on mechanism was the absence of any accumulation in serum of endotoxin bound to HDL as had been reported in studies of reconstituted HDL in endotoxemia (22). Lower serum endotoxin concentrations could be due to either faster clearance of free and lipoprotein-bound endotoxin or slower entry from the peritoneum to the plasma compartment. Although it cannot be excluded that PRE treatment reduced endotoxin input by blunting hypotension and bowel ischemia, reducing translocation of bacteria and endotoxin from the gut reservoir, we believe it is more likely that treatment with PRE enhanced removal of endotoxin from the circulation.

Serum endotoxin is cleared by the liver and excreted into the bile (2, 17, 26). Infusion of a triglyceride-rich, apolipoprotein-E-containing emulsion has been shown to direct hepatic endotoxin uptake away from Kupffer cells to parenchymal cells and out into bile (27). The combination of PL and cholic acid, used in PRE, may accomplish a similar redirection of endotoxin through the liver. HDL PL and cholesterol are cleared into the liver via hepatic scavenger receptor BI (SR-BI) and then excreted into bile (11, 13, 31). Intravenous infusion of HDL stimulates SR-BI-mediated biliary secretion of HDL PL, and coadministration of cholic acid with HDL increases secretion further (25).

The small number of animals in each dose group of this study limited our ability to detect dose-dependent differences in measures with high variability, such as serum TNF and endotoxin levels as well as many cardiopulmonary parameters. Two-way ANOVA comparing vehicle vs. combined treatment groups revealed a significant effect of treatment in TNF and several cardiovascular variables (Fig. 2). However, this was underpowered to detect difference between vehicle and multiple-dose groups.

Limitations of Study

The primary endpoint of this study was survival in a fatal model of septic peritonitis. The number of animals studied to satisfy this primary endpoint was insufficient to support statistically meaningful multiple comparisons by dose group of secondary endpoints, such as cardiac output, vascular resistances, and circulating endotoxin and cytokine levels. Because of high methodological and biological variability of serum endotoxin and cytokine data, preliminary power calculations estimate that ~20 animals per group would be necessary to detect a PRE dose effect, if it exists. To detect a PRE dose effect would require a different study design in which lethality in placebo-treated animals was 50% or less. In this design, sufficient numbers of placebo-treated animals would be alive at 24, 48, and 72 h to compare with the surviving PRE-treated pigs. Thus analysis of secondary endpoints, although scientifically quite interesting and relevant, is beyond the proposed scope of the present study.

In conclusion, prophylactic administration of a protein-free, PL-rich lipid emulsion provided positive survival benefit in a near-fatal porcine model of septic peritonitis. PRE decreased serum endotoxin and TNF-alpha levels, preserved cardiopulmonary function, and increased survival in a dose- and concentration-dependent manner. Systemic PL concentrations at the time of clot implantation of ~150, 300, and 600 mg/dl PL achieved a 57%, 88%, and complete (100%) survival, respectively. PL-rich emulsion may prove beneficial in treatment of hospitalized patients where serum cholesterol and PL levels are frequently low, suggesting clinical studies of PRE are warranted.


    ACKNOWLEDGEMENTS

The authors greatly appreciate the technical assistance of L. Phillips, S. Siddiqui, T. Balwani, E. Ribary, S. Qiu, Y. Chen, and J. Castillo-Robles. The authors thank A. Goldfarb for editorial assistance. We thank Roche Diagnostic Systems (Indianapolis, IN) for use of the COBAS FARA II.


    FOOTNOTES

Rush Heart Institute and The Rogosin Institute supported this project.

Preliminary portions of this study were presented at FASEB and Society of Critical Care Medicine meetings.

Address for reprint requests and other correspondence: R. D. Goldfarb, Section of Cardiology, Dept. of Medicine, Univ. of Medicine and Dentistry, New Jersey-Camden, 401 Haddon Ave, Suite 290, Camden, NJ 08103 (E-mail: roy.d.goldfarb{at}umdnj.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 24, 2002;10.1152/ajpregu.00285.2002

Received 21 May 2002; accepted in final form 22 October 2002.


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TOP
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INTRODUCTION
METHODS
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DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 284(2):R550-R557
0363-6119/03 $5.00 Copyright © 2003 the American Physiological Society



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