INFECTION AND IMMUNITY, Feb. 1992, p. 596-601 Vol. 60, No. 20019-9567/92/020596-06$02.00/0Copyright © 1992, American Society for Microbiology

In Vitro Inactivation of Bacterial Endotoxin by HumanLipoproteins and Apolipoproteins

KENNETH EMANCIPATOR,t* GYORGY CSAKO, AND RONALD J. ELINClinical Pathology Department, Warren G. Magnuson Clinical Center,

National Institutes of Health, Bethesda, Maryland 20892

Received 6 May 1991/Accepted 15 November 1991

A chromogenic Limulus amebocyte lysate assay was used to measure the recovery of 1 endotoxin unit ofendotoxin per ml. Purified human high-density lipoprotein, low-density lipoprotein, and apolipoprotein Al(apo Al) at a maximum concentration of 1 g of protein per liter reduced the recovery to <40% of baseline ina both dose- and time-dependent manner and in the absence of other serum components. Furthermore, thelapine fever response to a dose of 1 ml of 5-ng/mi endotoxin per kg was reduced by >0.5°C (P < 0.005) whenthe solution was preincubated in vitro with 0.5 g of apo Al per liter. By the Limulus test, a maximumconcentration of 0.01 g of apolipoprotein B (apo B) per liter (which contained deoxycholate, a knownendotoxin-disaggregating agent) reduced recovery to 0% in a dose- but not time-dependent manner. Inheat-inactivated (56°C, 1 h) normal human serum, high-density lipoprotein cholesterol (P < 0.005) and apo Al(P < 0.05) correlated inversely with endotoxin recovery, but, paradoxically, apo B correlated directly withendotoxin recovery (P < 0.05), while low-density lipoprotein cholesterol showed no significant correlation.INTRALIPID alone had no effect on endotoxin recovery. Addition of a maximum of 10 g of INTRALIPID perliter to 0.0042 g of apo B per liter increased endotoxin recovery from approximately 30 to 80% (P < 0.001),but addition of INTRALIPID to 0.25 g of apo Al per liter decreased recovery from approximately 30 to 20%(P < 0.001). We conclude that (i) lipoproteins are endotoxin inactivators; (ii) this ability of lipoproteins maybe modulated by their lipid component (lipid-endotoxin interaction); (iii) apo Al is capable of directlyinactivating endotoxin (protein-endotoxin interaction).

Plasma lipoproteins may interact with bacterial endotoxinand reduce its toxic activity. Murine and lapine high-densitylipoproteins (HDL) have been shown to bind endotoxin (11,13, 19, 22). Others reported that all major lipoprotein classesbind endotoxin in direct proportion to their plasma choles-terol concentration (in humans most of the cholesterol is inlow-density lipoprotein [LDL]) and concluded that a lipid-lipid interaction accounts for this binding (23). Incubation ofendotoxin with rabbit serum altered the chemical propertiesof the endotoxin and reduced its ability to induce a pyrogenicresponse and neutropenia in rabbits, its lethal effect inadrenalectomized mice, and its ability to activate lapinecomplement in vitro (20). LDL has also been described toprotect endothelial cells from the acute toxicity of endo-toxin, apparently because of the formation of endotoxin-LDL complexes (15). In addition, LDL, very-low-densitylipoprotein, chylomicrons, and HDL all have been observedto reduce the lethal effect of endotoxin in mice (8). Whilepreincubation of endotoxin with either delipidated rabbitplasma or human HDL alone did not reduce the pyrogenicityof endotoxin in rabbits, preincubation with both componentsdid reduce pyrogenicity (21). In turn, neither LDL norvery-low-density lipoprotein, when incubated with delipi-dated plasma and endotoxin, diminished the pyrogenic re-sponse of rabbits (21). It was proposed that inactivation ofbacterial endotoxin requires at least two steps, the firstinvolving disaggregation of endotoxin by an unidentifiedserum factor and the second involving binding of endotoxinto HDL (21).

* Corresponding author.t Present address: HFB-930, Division of Transfusion Science,

Center for Biologics Evaluation and Research, Food and DrugAdministration, 8800 Rockville Pike, Bethesda, MD 20892.

In the studies summarized above (8, 11, 13, 15, 19-23),endotoxin was used in microgram-per-milliliter concentra-tions. In clinically important situations, however, the con-centration of endotoxin in serum is probably in the picogramto nanogram per milliliter range. For instance, intravenousdoses of less than 4 ng of endotoxin per kg of body weightproduced signs and symptoms of endotoxemia in humanvolunteers (3). Assuming that intravenously injected endo-toxin is confined to the intravascular space and that theintravascular volume is 40 ml/kg of body weight, the result-ant concentrations of endotoxin in serum are less than 1ng/ml. Corollary to this observation is that circulating endo-toxin concentrations usually are less than 1 to 5 ng/ml inpatients with gram-negative sepsis (5, 9). The Limulus ame-bocyte lysate (LAL) assay has been recently used to deter-mine the capacity of normal human plasma to inactivateendotoxin in nanogram concentrations (25). This study,however, did not specifically address the role of lipopro-teins. More recently, normal human serum has been shownto inactivate nanogram quantities of endotoxin in anothermodel based on the release of monokines by monocytes andmacrophages (6). A possible role for lipoproteins was sug-gested by the observation that lipoprotein-depleted humanserum lacked the inhibitory capacity (6).Thus, despite numerous studies, the mechanism of inter-

action between endotoxin and plasma lipoproteins, as wellas the biological role and consequence of this interaction,remains poorly understood and controversial. We pursuedthese issues by studying the ability of human lipoproteinsand apolipoproteins to detoxify endotoxin for the highlysensitive LAL assay. Because LAL activity may not reflectthe toxicity of endotoxin in all circumstances, we also usedthe rabbit pyrogenicity test to demonstrate the endotoxininhibitory action of apolipoprotein Al (apo Al).

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MATERIALS AND METHODS

Sources of lipoproteins and lipid. We purchased HDL (20 gof protein per liter in 150 mM saline-0.01% EDTA, pH 7.4),LDL (5 g of protein per liter in 150 mM saline-0.01% EDTA,pH 7.4), apo Al (0.5 mg lyophilized without buffer salts), andapo B (0.5 mg lyophilized from 10 mM sodium deoxycho-late-50 mM NaCI-50 mM Na2CO3) from the BioneticsResearch Institute (Rockville, Md.). In the preparatoryprocess, HDL and LDL were washed with CsCl at the upperdensity limits of 1.21 and 1.063 g/ml, respectively, to removehigh-density (>1.21-g/ml) serum components. This high-density (>1.21-g/ml) serum fraction has been shown tocontain a component(s) that disaggregates endotoxin (7).Using lipoprotein electrophoresis with fat red 7B staining,both HDL and LDL were shown to be free of contaminationby other lipoprotein classes. HDL and LDL were deliveredto our laboratory within 1 day of purification and were usedno later than 2 weeks after purification. apo Al and apo Bwere >98% pure by scanning densitometry of Coomassieblue-stained sodium dodecyl sulfate-polyacrylamide gelelectrophoresis plates. The above-described lipoprotein-pro-tein materials as used in this study were negative with theLAL assay (see below).We reconstituted apo Al in 0.25 ml of pyrogen-free

phosphate (6.7 mM)-buffered saline (150 mM) containing0.01% EDTA (pH 7.4) (PBS-EDTA) and apo B in 0.5 ml of0.1 N pyrogen-free HCI (Sigma Chemical Co., St. Louis,Mo.). We verified the pH of each solution to be between 7.35and 7.45 before use. To verify pH without contaminating thesolution with endotoxin, we washed, after calibration, thepH electrode with 10 g of E-TOXA-CLEAN (Sigma Chem-ical Co.) per liter in pyrogen-free water and rinsed withpyrogen-free water. As a source of free lipid, we usedINTRALIPID 20% (KabiVitrum Inc., Alameda, Calif.),which consists of 200 g of soybean oil per liter, 12 g ofphospholipids per liter, and 22.5 g of glycerin per liter.Throughout the study, concentrations of INTRALIPID referto its soybean oil content.LAL assay. The LAL assay is based on the ability of

endotoxin to activate a system of proenzymes in the lysate(26). For endotoxin recovery studies, we used a chromoge-nic LAL assay (QCL-1000; Whittaker M. A. Bioproducts,Walkersville, Md.). In this method, the activated enzymeshydrolyze a peptide from para-nitroaniline with a resultantyellow color, measurable at 405 nm. The endotoxin added tosamples was the Escherichia coli O111:B4 endotoxin sup-plied with these kits. All pipette tips and glassware used inthe assay were autoclaved at 121°C for 16 h. Assays wereperformed according to the manufacturer's instructions ex-cept for preparation of standards (see below). Briefly, theassay is performed by adding 50 RI1 ofLAL to 50 pul of sampleand incubating at 37°C. After 10 min, 100 RI of chromogenicsubstrate is added. After an additional 6 min of incubation,the reaction is terminated by adding 100 pul of 50% (vol/vol)acetic acid. The A405 of the sample is corrected for sampleand reagent blanks, and the endotoxin concentration isdetermined from a standard curve. In some experiments,chloroform extraction of specimens was performed as pre-viously described (2).To calculate endotoxin recovery in samples containing

INTRALIPID, it was necessary to correct for lipemia.Therefore, we empirically derived corrected absorbancecurves. These curves relate the absorbance of fully devel-oped chromogen in PBS-EDTA containing a determinedquantity of INTRALIPID to the (corrected) absorbance of

that same amount of chromogen in PBS-EDTA withoutINTRALIPID. Endotoxin recovery was calculated from thiscorrected absorbance and the standard curve.

Blood samples. Blood samples from 45 healthy humandonors were drawn in pyrogen-free phlebotomy tubes (125-by 16-mm glass Vacutainer SST tubes [Becton Dickinson,Rutherford, N.J.] autoclaved at 121°C for 16 h). Sampleswere allowed to clot at room temperature for 30 to 60 minbefore separation of serum. Aliquots of serum from eachdonor were (i) sent to a reference laboratory (SmithKlineBio-Sciences Laboratories, Philadelphia, Pa.) for assay ofHDL cholesterol (by dextran sulfate precipitation and thenenzymatic assay) and apo Al and apo B (by rate nephelom-etry); (ii) assayed for triglycerides by a glycerol-blankedmethod with a COBAS FARA (Roche Diagnostics, Mont-clair, N.J.), using reagents from Behring Diagnostics, Inc.(Somerville, N.J.); and (iii) assayed for total cholesterol,albumin, total protein, calcium, magnesium, and phosphoruswith a Hitachi 736 with reagents supplied by BoehringerMannheim Diagnostics, Indianapolis, Ind. LDL cholesterolwas calculated (14). The remaining serum was heated at 56°Cfor 1 h to inactivate complement. To measure endotoxinrecovery, we added E. coli O111:B4 endotoxin and pyrogen-free water to produce a final concentration of 1 endotoxinunit (EU) per ml in a 1:10 dilution of complement-inactivatedserum. One endotoxin unit is defined as the biologicalactivity (by LAL assay) of 0.1 ng of U.S. Reference Stan-dard Endotoxin (RSE; E. coli 0113:HlO:K-negative, lotnumber EC-5; Center for Biologics Evaluation and Re-search, Food and Drug Administration, Bethesda, Md.).Standards were prepared by diluting endotoxin in pyrogen-free water. We measured endotoxin recovery after incubat-ing samples and standards at 37°C for 2 h.

Pyrogenic response of endotoxin in rabbits. We injectedrabbits with either endotoxin alone or a mixture of endotoxinand apo Al. To 4 ml ofRSE in water (100 EU/ml), we addedeither 4 ml of PBS-EDTA containing 1 g of apo Al per literor 4 ml of PBS-EDTA without apo Al. Each solution wasincubated at 37°C for either 12 or 16 h. Each of the foursamples (endotoxin with or without apo Al, incubated for 12or 16 h) was injected into three rabbits. The intravenous doseof endotoxin was adjusted for the animals' body weight; adose of 1 ml/kg was given. Rectal temperatures were re-corded just before injection and every 0.5 h after injectionfor a total of 3 h. For each rabbit, we calculated themaximum temperature, the maximum increase in tempera-ture above the preinjection baseline, and the fever index forthe 3 h after injection. The fever index is the area under thetemperature-versus-time curve, calculated by the trapezoi-dal rule, using the preinjection temperature as a baseline.

Statistical analysis. Regression analyses were performedwith StatView 512+ (BrainPower Inc., Calabasas, Calif.).For stepwise regressions, the threshold of the F test toinclude a variable in the model was set at 4.000, and thethreshold to remove a variable was set at 3.996. We usedStudent's t test (for unpaired samples) to determine thestatistical significance of the effect of INTRALIPID onendotoxin inactivation by apolipoproteins and to determinethe statistical significance of the observed differences infever response in rabbits. We used a paired-sample t test todetermine the statistical significance of differences in endo-toxin recovery as a function of incubation time of endotoxinwith lipoproteins.

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Protein Concentration (g/L)FIG. 1. Effect of lipoproteins and apolipoproteins on endotoxin

recovery. Purified HDL, LDL, apo Al, and apo B each induce adose-dependent decrease in endotoxin recovery in a serum-freemedium. (apo B also contains deoxycholate.) The vertical lines withbars show ±2 standard errors. The dashed lines represent the meanconcentrations of apo Al and apo B in 45 normal subjects.

- HDL- LDL-0-- apo A

0 1 2 3Incubation Time (hours)

FIG. 2. Dependence of endotoxin recovery on the preincubationtime of endotoxin with lipoproteins and apo Al. Endotoxin recoveryafter incubation with HDL (P < 0.005), LDL (P < 0.005), or apo Al(P < 0.01) at a protein concentration of 1 g/liter for 3 h at 37°C issignificantly lower than the recovery measured immediately afterendotoxin is added. Recovery was independent of the time ofincubation of endotoxin with apo B at a concentration of 0.0083,0.0042, or 0.0021 g/liter (data not shown).

RESULTS

To determine whether human HDL, LDL, apo Al, andapo B are endotoxin inhibitors, we added 1 EU of endotoxinper ml to determined quantities of HDL, LDL, apo Al, apoB, and mixtures of HDL and LDL diluted in PBS-EDTA.Standards were prepared by diluting endotoxin in PBS-EDTA. We measured endotoxin recovery after incubatingsamples and standards at 37°C for 3 h in the LAL assay.Endotoxin recovery as a function of HDL, LDL, apo Al, orapo B concentrations (expressed in terms of protein content)is presented in Fig. 1. For comparison, the mean concentra-tions of apo Al and apo B in serum samples from 45 healthydonors are also included. The figure shows that purifiedhuman HDL, LDL, apo Al, and apo B all dose dependentlydecreased the endotoxin recovery in serum-free medium. Inexperiments in which HDL and LDL were combined, asimple additive effect on the decrease of endotoxin recoverywas demonstrated (data not shown).A decrease in endotoxin recovery may theoretically occur

because a substance interacts with the endotoxin, the LAL,or the chromogenic substrate. In an attempt to determinewhich of these interactions is most likely, we performed twovariations of the previous experiment. In one variation, weadded 1 EU of endotoxin per ml to HDL, LDL, or apo Alwith a protein concentration of 1 g/liter, or to apo B at aconcentration of 0.0083, 0.0042, or 0.0021 g/liter, and mea-sured endotoxin recovery immediately or after 1.5 or 3 h ofincubation at 37°C. Even when added without preincubationto the LAL assay (i.e., at 0 h), all four lipoproteins andapolipoproteins decreased (by 20 to 60%) endotoxin recov-ery. For HDL (P < 0.005), LDL (P < 0.005), and apo Al (P< 0.01), however, endotoxin recovery measured after a 3-hpreincubation was significantly less than that measuredimmediately after its addition; the magnitude of changeranged from 10 to 30% (Fig. 2). Increasing the incubationtime from 0 to 3 h, we were unable to demonstrate anydifference in endotoxin recovery for apo B (data not shown).

In the other variation, we prepared activated LAL byadding PBS-EDTA containing 2 EU of endotoxin per ml toan equal volume ofLAL and incubating it at 37°C for 20 min.We added 100 ,ul of activated LAL to 100 R1 of chromogenicsubstrate containing no protein (control), 0.5 g of apo Al per

liter, or 0.0042, 0.0021, or 0.0010 g of apo B per liter. Weincubated the mixture for 3, 5, or 7 min at 37°C beforestopping the reaction by adding 100 ,ul of50% (vol/vol) aceticacid. Neither apo Al nor apo B, when added to the chro-mogenic substrate used in the endotoxin assay, inhibited thedevelopment of color by activated LAL (data not shown).Because both apo Al and apo B inactivated endotoxin for

the LAL assay, we wondered if the addition of lipids couldmodify their inhibitory potency. We added 1 EU of endo-toxin per ml to various concentrations of INTRALIPID (as asource of free lipids such as triglycerides and phospholipidswhich is commonly administered to patients) or mixtures offixed concentrations of apolipoproteins (0.25 g of apo Al perliter or 0.0042 g of apo B per liter) and various concentra-tions of INTRALIPID, diluted in PBS-EDTA. The concen-trations of apo Al and apo B were chosen because eachresulted in approximately 30% recovery of endotoxin in theabsence of INTRALIPID, allowing the observation of eitheran increase or a decrease after additions. As shown in Fig. 3,increasing concentrations of INTRALIPID, in the absenceof apolipoproteins, did not alter endotoxin recovery. When

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FIG. 3. Effect of free lipid on endotoxin inactivation by apolipo-proteins. Although INTRALIPID alone does not alter endotoxinrecovery, INTRALIPID does modulate the apo Al- and apo B-in-duced changes in endotoxin recovery: it enhances the endotoxininhibition by apo Al (P < 0.001) but attenuates the apo B-mediatedinhibition (P < 0.001). The vertical lines with bars show ±2 standarderrors.

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TABLE 1. Regression data for sera obtained from 45 healthy human donors'

Dependent variable Independent variable(s) Probability Correlation Slope Interceptcoefficient

Simple linear regressionEndotoxin recovery (%) HDL cholesterol (mmol/liter) 0.0023 -0.44 -14.3 56Endotoxin recovery (%) Triglycerides (mmol/liter) 0.0116 0.37 6.90 29Endotoxin recovery (%) apo Al (g/liter) 0.0335 -0.32 -12.7 61Endotoxin recovery (%) apo B (g/liter) 0.0377 0.31 16.1 23Endotoxin recovery (%) LDL cholesterol (mmol/liter) 0.1518 0.22 2.71 27HDL cholesterol (mmol/liter) apo Al (g/liter) 0.0001 0.73 0.904 -0.35HDL cholesterol (mmol/liter) apo B (g/liter) 0.0226 -0.34 -0.542 1.78Endotoxin recovery (%) Total cholesterol (mmol/liter) 0.3758 0.14 1.61 29Endotoxin recovery (%) Total protein (g/liter) 0.4478 -0.12 -0.459 70Endotoxin recovery (%) Phosphorus (mmol/liter) 0.5703 -0.09 -7.10 48Endotoxin recovery (%) Calcium (mmol/liter) 0.7002 0.06 7.58 20Endotoxin recovery (%) Magnesium (mmol/liter) 0.8448 0.03 7.34 31apo B (g/liter) apo Al (g/liter) 0.9489 0.01 0.008 0.85

Two-variable modelHDL cholesterol (mmol/liter) apo Al (g/liter) 0.0001 0.81 0.909 0.12

apo B (g/liter) 0.0004 -0.554

Stepwise regression with 5 nominal variablesEndotoxin recovery (%) HDL cholesterol (mmol/liter) 0.0092 0.52 -12.1 47

Triglycerides (mmol/liter) 0.0470 5.17apo Al (g/liter) Excludedapo B (g/liter) ExcludedTotal cholesterol (mmol/liter) Excluded

a Endotoxin recovery was determined after a 2-h preincubation in 1:10 dilutions of donor serum samples and related to various other analytes. Statisticallysignificant (P < 0.05) correlations are shown in boldface.

added to apo Al, increasing concentrations of INTRALIPIDcaused a slight, but statistically significant (P < 0.001),decrease in endotoxin recovery. Finally, when added to apoB (containing deoxycholate), increasing concentrations ofINTRALIPID caused a statistically significant (P < 0.001)and numerically large increase in endotoxin recovery.The physical properties of plasma lipoproteins and apo-

lipoproteins may change substantially in an artificial matrix(i.e., buffer). EDTA is used to minimize these physicalchanges, but the EDTA itself may theoretically modify theobserved endotoxin recoveries. These possibilities prompt-ed us to study endotoxin recoveries in the absence of EDTAin human serum specimens that likely contain intact lipopro-teins and apolipoproteins. In addition, experiments withnormal human sera also allowed the study of possiblecorrelations between endotoxin recovery and the concentra-tions of various serum constituents. The mean ± standarddeviation concentrations of apo Al and apo B in 45 donorserum samples were 1.84 ± 0.35 and 0.86 ± 0.27 g/liter,respectively. The mean ± standard deviation recovery ofendotoxin in 1:10 dilutions of these serum samples was 37 +14% after a 2-h incubation at 37°C. Regression data relatingendotoxin recovery and the other serum analytes in donorserum are summarized in Table 1. The first section of Table1 shows that HDL cholesterol, triglycerides, apo Al, andapo B were the only analytes which had statistically signif-icant (P < 0.05) correlations with endotoxin recovery. In thesecond section of Table 1, a two-variable model demon-strates that there is a significant (P < 0.0004) inversecorrelation between HDL cholesterol and apo B, even aftercorrection for the direct correlation between HDL choles-terol and apo Al. This suggests that the positive correlationbetween endotoxin recovery and apo B results from acollinearity problem, simply reflecting the existence of in-verse correlation between HDL cholesterol and apo B. In

the third section of Table 1, the results of a stepwiseregression analysis are shown. The stepwise regressiondemonstrates that once the HDL cholesterol is known, apoAl and apo B do not significantly contribute to the predictionof endotoxin recovery in serum; only the serum triglycerideconcentration does, but with marginal significance (P <0.047).To corroborate the in vitro inactivation of endotoxin by an

assay system independent of LAL, and because pyrogenic-ity is a prominent biological action of endotoxin, we moni-tored the lapine fever response to intravenously injectedendotoxin with or without preincubation with apo Al. Table2 shows that the maximum temperature, the maximumincrease in temperature, and the fever index were all signif-icantly lower when apo Al was preincubated with endotoxinfor 12 to 16 h at 37°C before injection. We also did recoverystudies from the preparations used for intravenous injectionsin the LAL assay. The high (>90%) recovery of endotoxinby chloroform extraction from the RSE-plus-apo Al mixtureindicated that the reduced pyrogenicity was due to a specificinteraction between apo Al and endotoxin (protein-endo-toxin interaction) and was not caused by adsorption ofendotoxin to the container or other nonspecific mechanisms(Table 2).

DISCUSSION

In this work, we demonstrated that purified human HDL,LDL, and apo Al each decrease in vitro the endotoxinrecovery in the absence of other serum factors. apo B (whichcontains deoxycholate) also decreased the recovery of en-dotoxin. Furthermore, we observed that the mean proteinconcentrations of apo Al and apo B in healthy humanvolunteers are greater than that needed for inactivation ofendotoxin.

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TABLE 2. Effect of apo Al on the pyrogenic response of rabbits to endotoxin

Pyrogenic response in rabbits"' % Endotoxin recovery by LALbSample Maximum temp ('C) Maximum change in Fever index (h 'C) No Chloroform

(mean ± SE) temp (°C) (mean ± SE) (mean ± SE) extraction extraction

RSE 41.25 ± 0.062 1.683 ± 0.060 3.142 ± 0.147 100, 98 92, 95RSE + apo Al 40.65 ± 0.138 1.117 ± 0.164 1.917 ± 0.323 23, 22 92, 94

Probability 0.0014 0.0044 0.0031

a Rabbits (six per group) were injected with a 1-ml/kg dose of either a solution containing 50 EU of RSE per ml or a solution containing 0.5 g of apo Al perliter in addition to 50 EU of RSE per ml (preincubated for 12 to 16 h at 37°C). Results of endotoxin recovery from these preparations using the LAL assay alsoare included.

b 12-h incubation, 16-h incubation.

In theory, the apparent inactivation of endotoxin couldoccur because lipoproteins bind either endotoxin or one ormore of the reagents used in the assay for endotoxin. Wehave shown that neither apo Al nor apo B inhibits thehydrolysis of the chromogen by activated LAL. Therefore,either lipoproteins must inactivate endotoxin or they mustinactivate one or more of the proenzymes in the LALcascade, preventing the formation of active enzymes. Twoobservations favor the former conclusion. First, endotoxinrecovery decreased with increasing incubation time of endo-toxin with HDL, LDL, or apo Al. Because this incubationoccurs before any LAL reagents are added, the decreasingrecovery with increasing incubation time is most compatiblewith a direct interaction between endotoxin and lipopro-teins. Second, the pyrogenic response in rabbits decreased ifendotoxin was incubated with apo Al. Similar evidence ledother investigators to favor the conclusion that whole lapineserum inactivates endotoxin, rather than LAL (24, 25). It isdifficult, if not impossible, to completely exclude the possi-bility that lipoproteins also partially inactivate LAL. How-ever, even if this phenomenon does occur, it does not negatethe evidence that lipoproteins inactivate endotoxin.Although previous studies (7, 23) suggested lipid-endo-

toxin interactions in the inactivation of endotoxin by lipo-proteins, our present results obtained with apo Al and apo Bindicate protein-endotoxin interactions in the detoxificationprocess. Three lines of evidence support a protein-endotoxininteraction. First, both apo Al and apo B, in the absence oflipid, reduced the functional activity of endotoxin, as as-sayed with LAL. Second, apo Al, in the absence of lipid,reduced the lapine pyrogenic response to endotoxin. Third,INTRALIPID alone did not reduce the functional activity ofendotoxin.

Ulevitch et al. (21, 22) have proposed a two-step mecha-nism for the formation of endotoxin-HDL complexes: in thefirst step, unidentified serum factors disaggregate endotoxin;in the second step, endotoxin binds with HDL. According tothis concept, endotoxin-HDL complexes do not form in theabsence of other serum factors. Our experiments support thefindings of Munford et al. (12) that endotoxin-HDL com-plexes may also form in the absence of disaggregatingagents. In the absence of any disaggregating agent, the LALactivity of endotoxin was decreased by HDL, LDL, or apoAl and the pyrogenic activity of endotoxin was decreased byapo Al. Because our studies were performed with patho-physiologically relevant concentrations of endotoxin andHDL, LDL, or apo Al proteins, there was 107 times as muchprotein as endotoxin by weight. This excess ofHDL or LDLprotein may explain why inactivation was observed in theabsence of disaggregating agents. Presumably, the excessprotein compensates for a very slow reaction rate. These

findings do not refute a synergistic role for disaggregatingagents in the formation of endotoxin-lipoprotein complexes.In fact, it seems quite likely that the observed potency andefficacy of apo B for endotoxin inactivation is a result of asynergistic action by deoxycholate. Deoxycholate has beenshown to augment the formation of endotoxin-HDL com-plexes (12).Having established that apo Al and apo B inactivate

endotoxin, we studied the effect of adding variable amountsof free lipids (INTRALIPID) to the reaction mixture. IN-TRALIPID contains virtually no cholesterol, but abundanttriglycerides; INTRALIPID is most similar to the lipidcomponents of very-low-density lipoprotein. This may ex-plain why INTRALIPID induced such dramatic changes inapo B-mediated endotoxin inhibition but comparativelysmall changes in the apo Al-mediated inhibition.Endotoxin recovery studies were performed with normal

human sera to rule out artifacts created by the presence ofEDTA (and other matrix effects) in experiments with iso-lated lipoproteins. As expected, there was a negative corre-lation between endotoxin recovery and either apo Al orHDL cholesterol. Remarkably, HDL cholesterol was abetter predictor of endotoxin recovery than was apo Al.This conflicts with the observation that apo Al appears to bea more potent endotoxin inhibitor in serum-free medium, butcorroborates the observation of a statistically significant(albeit quantitatively small) enhancement of apo Al-medi-ated inhibition by the addition of INTRALIPID. apo A2, Cl,C2, C3, D, and E, which are also components of HDL (16),may also play a role.The positive correlation between endotoxin recovery and

apo B in human sera is quite unexpected, given that apo Bappears to inhibit endotoxin activity. This positive correla-tion is most likely an epiphenomenon due to a colinearityartifact in the regression data: in this study, as well as in alarger study (4), a negative correlation between apo B andHDL cholesterol was observed. Therefore, the positivecorrelation between endotoxin and apo B does not rule out arole for apo B to inhibit endotoxin in human serum. In fact,other investigators (1) have observed a statistically signifi-cant negative correlation between apo B and endotoxinrecovery, under very similar experimental conditions. Thepositive correlation observed between concentration of tri-glycerides in serum and endotoxin recovery suggests thattriglycerides attenuate some mechanism of endotoxin inhi-bition. apo B is a likely candidate given that the triglyceride-rich INTRALIPID attenuated apo B-mediated endotoxininhibition.Because the matrix of the LAL assay was not as uniform

with donor serum as it was with serum-free medium, the

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finding that endotoxin recovery is not related to the concen-trations of calcium, magnesium, phosphorus, or albumin isimportant in that it excludes possible sources of artifact.Activation of LAL depends on calcium and magnesiumconcentrations (18). The ionic activity of these analytes isaltered by phosphorus and albumin. Proteins in general donot appear to be endotoxin inhibitors because endotoxinrecovery was unrelated to albumin or total protein concen-trations.The present study has addressed in vitro inhibition of the

activity of endotoxin. While this effect would seem to bebeneficial pathophysiologically, binding of endotoxin to lipo-proteins may also have some untoward biological effects.For example, it has been suggested that binding of endotoxinto HDL contributes to organ damage such as adrenal hem-orrhage by facilitating transport of endotoxin into cells byapo E-mediated mechanisms (10). While most studies (11-13, 19-22) primarily implicate HDL in the binding andinactivation of endotoxin, some studies (6, 15, 23) suggestthat LDL is more effective in this regard. It is interesting tocomment on the role of lipoproteins in endotoxin inactiva-tion with respect to the recently described lipopolysaccha-ride (LPS, endotoxin)-binding protein (17). LPS-bindingprotein binds to the lipid A moiety of LPS and, forminghigh-affinity complexes with LPS that bind to monocytes andmacrophages, appears to control the response to LPS underphysiological conditions (17). It was proposed that "a prin-cipal function of LBP [LPS-binding protein] may be toenhance the ability of the host to detect LPS early ininfection; release of cellular products would then enhancenatural resistance mechanisms that combat infection" (17).We surmise that plasma lipoproteins may represent anotherline of defense in neutralization of endotoxin when infectionsprogress beyond the initial stage. The action of various hostfactors induced by endotoxin in the presence of plasmalipoproteins requires further study.

ACKNOWLEDGMENT

We thank Donald Hochstein, Center for Biologics Evaluation andResearch, Food and Drug Administration, Bethesda, Md., forperforming the pyrogen testing in rabbits.

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