Lipoprotein Abnormalities Associated with Lipopolysaccharideinduced Lecithin:Cholesterol Acyltransferase and Lipase Deficiency*

Transcription

1 THE JOURNAL OF BOLOGCAL CHEMSTRY by Te American Society for Biocemistry and Molecular Biology, nc Vol. 264, No. 17, ssue of June 15, pp ,1989 Printed in U. S. A. Lipoprotein Abnormalities Associated wit Lipopolysaccarideinduced Lecitin:Colesterol Acyltransferase and Lipase Deficiency* Bruce J. AuerbacS and Jon S. Parks5 (Received for publication, August 3, 1988) From te Department of Comparatiue Medicine. Bowman Gray Scool of Medicine, Wake Forest University Medical Center, Winston-Salem, Nort Carolina Density gradient ultracentrifugation was used to iso- tween acute and cronic inflammatory events and plasma late and caracterize te plasma lipoproteins from Af- lipid concentrations (1-5). Canges in plasma lipids ave rican green monkeys before and and after been associated wit bacterial, viral, and protozoal infections subcutaneous injection of 300 pg/kg lipopolysaccaride as well as cancer (1, 4, 6-8). Te most typical response to (LPS) to induce an acute pase response. Compared infection is elevated plasma triglycerides wit a decrease in wit 0 values, reductions occurred in plasma colesterol (39%), ig density lipoprotein (HDL) colesterol total plasma and HDL colesterol concentrations. Te ac- (54%), 1ecitin:colesterol acyltransferase (LCAT) acquired ypocolesterolemia tat accompanies infections or tivity (55%), and post-eparin plasma lipase activity malignant illness as even been suggested as an indicator of (68%) after LPS injection wile plasma triglyc- poor prognostic outcome (9). eride concentrations increased 700%. Colesterol dis- Probably te best studied experimental procedure of te tribution among lipoproteins sifted from 7 to 41% in interrelationsip between infection and ost response as very low density lipoproteins (VLDL), 65 to 38% in been te injection of lipopolysaccaride (LPS) in experimenlow density lipoproteins (LDL), and 28 to 21 % in HDL tal animals. LPS is a complex macromolecule, containing after LPS injection. At after LPS injection, all lipid and polysaccarides, tat is present in te cell wall of lipoprotein classes were relatively enriced in pos- Gram-negative bacteria (10). Wen LPS is injected into expolipid and triglyceride and depleted of colesteryl perimental animals or umans in subletal doses, it results ester. Te plasma concentration of all cemical con- in a complicated series of patological responses including stituents in VLDL was increased 3-%fold witin fever, leukocytosis, disseminated intravascular coagulation, after LPS injection. By negative stain electron micros- and te increased syntesis and secretion into plasma of a copy, HDL were discoidal in sape wile VLDL and class of proteins collectively referred to as acute pase reac- LDL appeared to ave excess surface material present. tant proteins (10, 11). Wen macropages encounter LPS Even toug total HDL protein concentration in plasma was unaffected, te plasma mass of te smallest tey are stimulated to secrete a number of monokines includ- HDL subfractions (HDL3,,,,) doubled wile te mass of ing interleukin-1 and cacectin tat cause many of te obintermediate-sized subfractions (HDL3,) was dramati- served systemic effects of infection. Wit regard to te effects cally decreased witin after treatment. HDL became enriced in apoe, acquired aposaa, and became depleted of apoa-, A-11, and Cs by after LPS injection wile apob- 100 remained te major apoprotein of VLDL and LDL. We conclude tat administration of LPS to monkeys prevents normal intravascular metabolism of lipoproteins and results in te accumulation of relatively nascent forms of lipoproteins in plasma. Tese immature lipoproteins resemble tose isolated from te recirculating perfusion of African green monkey livers, wic are relatively deficient of LCAT activity and tose isolated from te plasma of patients wit familial LCAT deficiency. Recent studies ave demonstrated an interrelationsip be- * Tis researc was supported by National Heart, Lung, and Blood nstitute Grants HL (Specialized Center of Researc in Arteriosclerosis), HL-736, HL-38066, and HL Te costs of publication of tis article were defrayed in part by te payment of page carges. Tis article must terefore be ereby marked aduertisement in accordance wit 18 U.S.C. Section 1734 solely to indicate tis fact. $ Tis work was carried out as partial requirement for te Master of Science degree in Comparative and Experimental Patology from te Bowman Gray Scool of Medicine of Wake Forest University. 0 To wom correspondence and reprint requests sould be addressed on lipid concentrations, bot interleukin-1 and cacectin inibit lipoprotein lipase activity in uitro, and cacectin as been sown to inibit tis enzyme in vivo (12-14). Lipoprotein lipase is a key enzyme in te ydrolysis of plasma triglyceride, and its inibition is tougt to lead to te observed ypertriglyceridemia wen LPS is given to experimental animals. Te cause of te ypocolesterolemia seen wit infections or observed after LPS administration is poorly understood. Altoug te effects of LPS on plasma lipid concentrations are well known, tere is little information regarding te effect of LPS on plasma lipoprotein composition and distribution. Tis study was undertaken to caracterize te canges in lipoprotein composition, distribution, and concentration wen LPS was given to African green monkeys. We ave used tis species in te past to study te effects of environment factors on lipoprotein caracteristics (15, 16). African green monkeys fed diets typical of tose consumed by Nort Americans ave lipoprotein profiles more similar to tat of man tan many oter nonuman primate species (17). We found tat LPS administration to monkeys appears to prevent normal intravascular metabolism of lipoproteins and results in te accumulation of immature forms of lipoproteins tat Te abbreviations used are: HDL, ig density lipoprotein; LCAT, lecitin; colesterol acyltransferase; LPS, lipopolysaccaride; VLDL, very low density lipoprotein; SDS, sodium dodecyl sulfate; LPL, lipoprotein lipase; LDL, low density lipoprotein; aposaa, serum amyloid A protein.

2 resemble tose isolated from monkey liver perfusates or te plasma of uman beings wit familial LCAT deficiency. MATERALS AND METHODS Experimental Animals-Eigt adult (4 male/4 female) African green monkeys (Cercopitecus aetiops), weiging kg,were used in tis study. Te animals were maintained on a diet containing 0.78 mgof colesterol/kcal and 40% of calories as fat (butter or safflower oil) for a period of at least 3 years before te study began (18). Te animals were generously supplied by Dr. Lawrence L. Rudel, Department of Comparative Medicine of Te Bowman Gray Scool of Medicine. Lipoprotein solations-all animals were fasted at least 18 prior to blood samplings or administration of LPS. A solution of LPS (2.5 mg/ml) from Escericia coli, serotype 055:B5 (Sigma) was prepared and injected subcutaneously in te abdomen at a dose of 300 pg/kg bodyweigt into animals sedated wit 10 mgof Ketamine HCL (Bristol Laboratories, NY)/kg body weigt. Control animals were given injections of saline. At te time of blood sampling, te fasted animals were sedated wit Ketamine, and blood was collected in tubes containing a final concentration of 0.1% EDTA, 0.04% 5,5'-ditiobis(nitrobenzoic acid), and 0.01% sodium azide, ph 7.4. Red blood cells were removed by centrifugation at 39,000 X g/min. Lipoproteins were ten isolated from te plasma by adjusting te plasma solvent density to g/ ml by te addition of solid KBr, followed by centrifugation of plasma for 20 at 50,000 rpm at 4 "C in a Ti-50 rotor (Beckman nstruments, nc., Palo Alto, CA). Te lipoproteins were removed by slicing te top 1 cm of te tube and aspirating te supernatant. Density gradient centrifugation was used for lipoprotein class fractionation. A discontinuous gradient was establised by underlaying 4.5 ml of 1.006, 5.5 ml of 1.030, and 2.5 ml of g/ml KBr solutions in SW 40 rotor tubes (Beckman nstruments). Te KBr solutions were made by addition of solid KBr to 0.9% NaC1, 0.01% EDTA, 0.01% sodium azide. Te lipoprotein samples were dialyzed to a density of g/ml and added to te d = g/ml segment of te gradient. Te tubes were centrifuged at 40,000 rpm for 40 at 4 "C in an SW 40 rotor. Te gradients were drained out of te top of te tubes troug a UV monitor after piercing te bottom of te centrifuge tube and pumping in a dense (1.9 g/ml) solution of Fluorinert (3M Company, St. Paul, MN). Fractions of 0.35 ml were collected using a fraction collector. Te density profile of te samples was determined by a standard curve generated by reading te refractive index of solutions of known densities tat were prepared by te addition of solid KBr to saline. Fractions containing discrete peaks were pooled for furter analysis. Fractions pooled from te density gradient run on te - and - plasma samples were cosen using te 0 plasma sample for eac animal as te reference, suc tat, for eac animal equivalent density gradient fractions of 0-, -, and - plasma samples were taken for analysis. Plasma HDL colesterol concentrations were also determined using te eparin-manganese precipitation procedure (19). Electron Microscopy-solated lipoprotein fractions, at a concentration of 2 mgof total mass/ml, were negatively stained wit 2% potassium pospotungstate, ph 6.5, on Formvar-carbon-coated grids. Te negatively stained lipoproteins were observed wit a Pilips 400 transmission electron microscope (Pilips Electronic nstruments, Mawa, NJ). Cemical Analyses-Cemical composition of te isolated lipoproteins was determined as follows. Aliquots of lipoproteins were lyopilized and were extracted wit cloroform/metanol (2:l). Te extracted lipids were dried under nitrogen and weredissolved wit cloroform. Approximately 300pgof total lipid were applied to activated silica gel plates, wic were developed using a solvent system of exane/etyl eter/acetic acid (80:202 v/v/v). Te separated lipids were scraped from te plate and eluted from te silica gel wit eiter cloroform (free colesterol and colesteryl ester) or cloroform/metanol (2:l for triglyceride). Concentrations of free colesterol and esterified colesterol were determined according to te metod of Rudel and Morris (20). Plasma colesterol and total colesterol concentrations of aqueous aliquots of isolated lipoproteins were also measured by an enzymatic metod (Gilford Single Vial Reagent System, Gilford Diagnostics, Cleveland, OH). Protein content was measured by te metod of Lowry et al. (21), after color development and extraction of te samples wit exane. Pospolipid posporus was measured by te metod of Fiske and SubbaRow (22). Triglycerides isolated by tin layer cromatograpy were quan- LPS-induced Lipoprotein Canges titated by te metod of Sardesai and Manning (23). Plasma triglycerides were also quantitated by tis metod after adsorption of plasma pospolipids wit activated Florisil (). Gradient Gel Electroporesis-Prepoured gradient gels (4-30%; Parmacia LKB Biotecnology nc.) were used to investigate lipoprotein subfraction size eterogeneity as described previously (25, 26). Briefly, d < g/ml lipoprotein (four parts) were mixed wit 1 part of a solution consisting of 40% sucrose and 0.01% brompenol blue; a pl aliquot containing 10 pg of protein was applied to te gels. Gels were subjected to electroporesis for at 125 V (10 "C) and after electroporesis gels were stained wit Coomassie Blue G-250. After destaining, te gels were scanned using a laser densitometer. Te relative migration distance (RF) for an individual band for eac sample was calculated by taking te ratio of te migration distance of te band relative to te migration distance of bovine serum albumin in te standard lane of te same gel. Eac peak was designated as eiter HDLz (RF = , HDL3, (RF = ) or HDL3b,e (RF = ) based on te nomenclature of Blance et al. (25) and te area under eac peak was calculated by dropping vertical lines from te troug of eac subfraction peak to te abscissa. Plasma concentration of individual HDL subfractions was calculated by multiplying te percentage protein distribution derived from gradient gel electroporesis by te concentration of total HDL protein in plasma. Te latter was calculated from te Lowry protein concentration of te density gradient isolated HDL and was corrected back to wole plasma concentration by colesterol recovery. SDS-Gradient Gel Electroporesis-Qualitative analysis of apopro- teins was done using sodium dodecyl sulfate (SDS)-gradient gel electroporesis. Prepoured gels (4-30%) were equilibrated wit SDS by prerunning te gels for 2 at 100 V (15 "C) wit an electrode buffer consisting of 50 mm Tris-HC1, 20 mm sodium acetate, 2 mm EDTA, 0.2% SDS, ph 8.6. Samples were dialyzed against 0.01% EDTA, 0.01% NaN3, ph 7.4, and were ten lyopilized. A solution containing 1% SDS, 8% sucrose, and 0.002% brompenol blue was added to te sample to give a final protein concentration of 1 mg/ml. Aliquots of samples containing 5-10 pg of protein were eated to 100 "C for 2 min and were subjected to electroporesis of 4 at 100 V (10 "C). After electroporesis, gelswere stained overnigt wit 0.2% Coomassie Blue R-250 in 50% metanol, 10% acetic acid and were destained consecutively wit 50% metanol, 10% acetic acid and 10% metanol, 10% acetic acid. Enzyme Assays-Plasma LCAT activity was measured by te addition of a radiolabeled artificial substrate to plasma using a modification (27) of te procedure of Cen and Albers (28). Briefly, recombinant particles consisting of egg yolk lecitin, ["C]colesterol, and uman apoa-1 (250:80.8 molar ratio) were made by te colate dialysis metod. Eac assay contained 30 p1 of plasma, 3 pgof substrate colesterol, 2% uman serum albumin, and 5 in a final volume of 495 pl. Te samples were incubated in a saking water bat at 37 "C for 30 min. Te incubation was stopped by te addition of 495 p1 of etanol. Te lipids were extracted twice wit 5 and 3 ml of exane containing 20 pg/ml eac of colesteryl oleate and free colesterol as carriers. Te extract was ten dried under a stream of nitrogen and redissolved in cloroform. Te lipids were ten separated by tin layer cromatograpy as described previously, te CE spot was scraped, and te radioactivity was measured in a liquid scintillation counter. LCAT activity was reported as nanomoles of colesteryl ester formed/milliliter of plasma/our. Post eparin plasma triglyceride lipase activity was measured using ntralipid (Cutter Laboratories, Berkely, CA) containing tri(9:lo- 3H)oleoylglycerol as te substrate (46). Post-eparin plasma was obtained 20 min after te intravenous injection of 100 units of eparin/kg body weigt. Ten p1 of post-eparin plasma was added to 90 p1 of te substrate solution containing te labeled ntralipid and 1% albumin in 0.2 M Tris buffer, ph 8.5. HDL was added to provide a source of apo-c, an LPL activator. Te mixture was incubated at 37 "C for 60 min, and te reaction was stopped wit te addition of 1.5 ml of benzene/cloroform/metanol (1:0.5:1.2, v/v/v) containing 0.1 mm oleic acid as carrier. A 0.3-ml aliquot of 0.3 N NaOH was ten added to te solution, te mixture was vortexed, and te pases were separated by low speed centrifugation. An aliquot of te upper pase was taken, and te radioactivity was determined by liquid scintillation counting. One unit of activity was equal to 1.0 pmol of oleic acid ydrolyzed/. Statistical Analysis-All values are given as mean & S.E. Statistical comparisons of base-line values to and post-injection values weremade using repeated measures analysis of variance and te Fiser's least significant difference test (29).

3 Lip( LPS-induced oprotein Canges a RESULTS Plasma Colesterol,Triglyceride, and HDL Concentrations"Bacteria1 endotoxin was injected subcutaneously in te abdomen of eigt African green monkeys at a dose of 300 pgl kg body weigt to study its effect on plasma lipid concentrations. During preliminary studies, tis dose did not result in a measurable increase in body temperature at te indicated time points nor in any clinical signs of illness of te animals. Fig. 1 sows te concentrations of total plasma and HDL colesterol and plasma triglyceride at and after injection. Te total plasma colesterol concentrations decreased 33% ( p < 0.001), HDL concentrations (HDL-C) fell 21% ( p < 0.04),and triglyceride concentrations were sligtly iger after LPS injection. Forty-eigt after injection total plasma colesterol and HDL-C concentrations were still significantly ( p < 0.02) lower (29and 54%, respectively) tan preinjection concentrations wile plasma triglycerides were 7-fold iger (20 f 1uersus 136 f 14 mg/dl; p < 0.015). Two animals were followed for a longer period of time; witin 72 afterlps injection te totalplasma colesterol, triglyceride, and HDL concentrations were approacing base-line values, but 6 days were required for all values to return to normal in tese two animals. n control studies two animals ad mean total plasma colesterol values of 429, 406, and 431 mg/dl at 0,, and after injection of saline. Te corresponding mean HDL-C values after normal saline injection were, 56, and mg/dl. Triglyceride concentrations were not determined for te control animals. To determine te effect of LPS administration on lipoprotein distribution, plasma lipoproteins were fractionated for six of te eigt animals using density gradient centrifugation and te colesterol distribution was measured. Generally, tree distinct regionswere apparent tat corresponded to VLDL, LDL, and HDL based on cemical compositions (Tables 11-V), apoprotein composition (Fig. 2), agarose gel mobility (data not sown), and size on gradient gels of tese isolated fractions. n four of te animals, te LDL region contained two peaks (designated as ligt and eavy LDL) 16.3 tat werepooled separately and analyzed. Te colesterol distribution of eac region fromte density gradient analysis is given in Table. At base-line LDL contained muc of te colesterol mass (65%)wit very little invldl (6.5%).After LPS administration te proportion of colesterol distributed in VLDL increased 6-fold relative to base line wile te proportion of colesterol in LDL decreased to 60% of te preinjection value. LipoproteinCompositions-Cemical compositions were measured on te lipoprotein fractions isolated by density PLASMA LPDS e a 100 " TPC HDL-C TG FG. 1. Effect of LPS injection (300 pg/kg) on plasma lipid concentrations. Total plasma colesterol (?'PC),HDL colesterol (HDL-C),and triglyceride ( X ) concentrations were determined for six to eigt animals at 0,, and after LPS injection. Values represent mean f S.E.Asterisk denotes values tat are significantly different ( p < 0.05) from te 0 by repeated measures analysis of variance and Fiser's least significant difference test. b c d s f a i i k m n!! i E!A SA A ;A '/c FG. 2. SDS-polyacrylamide gradient (4-30%) gel electroporesis of lipoprotein fractions (VLDL, LDL,and HDL) isolated by density gradient centrifugation. Ten pg of protein were pooled from eac fraction from four monkeys injected wit LPS. Details of electroporesis conditions are given under "Materials and Metods." Lanes a and n (low molecular weigt standards); lanes b-e = VLDL, LDL (ligt), LDL (eavy), HDL, respectively at 0 ; lanes f-i = VLDL, LDL (ligt), LDL (eavy), HDL, respectively, at after LPS injection and lanes j-m = VLDL, LDL (ligt), LDL (eavy), HDL, respectively, at after LPS injection. Apoprotein regions are indicated on rigt-and margin. TABLE Lipoprotein colesterol percentage m s distribution of Africangreen monkeys before and after administration of LPS Time after LPS injection?6 Colesterol distribution VLDL" LDL HDL * f f f4.2 f f8.9 f6.4 f7.3 VLDL = region, LDL = region 11, and HDL = region 11 of te density gradient profile sown in Fig. 2. bmean f S.E.( n = 6). gradient ultracentrifugation. Tables 11-V sow te results for VLDL, LDL, and HDL, respectively. All of te cemical constituents of VLDL sowed significant (p < 0.05) mass increases after LPS injection (Table 11).However, wen analyzed as percentage composition, te major canges in VLDL compositioninvolved an enricment in triglyceride and PL and a decrease in CE content. Te relative depletion of VLDL colesteryl ester was nearly equal to teenricment of VLDL triglyceride suc tat tepercentage of core neutral lipid was similar between 0 uersus time points. Te cemical compositions of LDL are sown in Table111. Twenty-four after LPS injection tere was a 53% decrease in plasma concentration of LDL protein ( p < 0.05) and a70% decrease in LDL colesteryl ester ( p < 0.05). Large canges in te percentage of LDL pospolipid (21 uersus 31%) and colesteryl ester (46 uersus 27%) were also observed between base-line and - plasma samples.two days after LPS injection tere was a 3-fold increase in LDL triglyceride concentration and a 74% reduction in LDL colesteryl ester suc tat te two neutral lipids were comparable in concen-

4 Time" TABLE 1 Cemical composition of plasma VLDL before and after LPS administration Protein mgldl * k1.9 k1.4 ko.9 k2.3 (11.9%)' (18.9%) (8.1%) (20.9%) k5.2 k3.4 f2.2 k5.9 (13.0%) (.0%) (21.7%) (9.7%) 39.3d 78.4d.7d 111.Od k f5.8 k44.6 (12.4%) (.8%) (7.8%) (35.1%) "Time after LPS injection. 'Mean k S.E. (n = 6). 'Mean % composition. versus p < TABLE 11 Cemical composition of plasma LDL before and after LPS administration Time" Protein Pospolipid c ~ ~ ~ Triglyceride ~ r o l ' (20.9%)' 42Ad k8.4 (21.0%) (21.5%) 90.2 k17.2 (21.4%) 64.6 k14.1 (31.6%) 97.7 k19.7 (34.3%) "Time after LPS injection. 'Mean k S.E. (n = 6). 'Mean % composition. versus ; p < '0 versus ; p < mgldl 36.5 k7.2 (8.7%) 25.4 f6.2 (12.4%) 30.3 k9.4 (10.6%) LPS-induced Lil poprotein Canges k2.4 (3.3%) 15.1 k5.4 (7.4%) 45.8' k10.5 (16.1%) TABLE V Cemical composition of plasma HDL before and after LPS administration Time" Protein Pospolipid colesterol Free ' 14.3 k33.0 (42.5%)' k29.1 (41.6%) f35.1 (3.7%) (31.8%) mddl k d 27.6d k32.9 f7.4 (37.2%) (7.1%) k34.9 f32.2 f2.8 (45.5%) (38.2%) (4.4%) "Time after LPS injection. 'Mean f S.E. (n = 6). 'Mean % composition. versus ; p < '0 versus ; p < k7.2 (40.3%).3 k7.0 (31.7%) 62.6d k13.7 (19.8%) Colesteryl ester k28.7 (45.7%) 56.4d k12.5 (27.6%) 49.9' k12.8 (17.5%) Triglyceride Colesteryl ester k18.8 (1.5%) (20.6%) d k4.3 k14.3 (2.4%) (11.6%) 14.6' 26.5' k4.9 k11.7 (4.2%) (7.7%) tration and percentage composition in plasma LDL. LDL protein concentration at was still 30% lower tan baseline values but tis difference was not statistically significant. Te percentage of LDL protein did not cange at eiter time point after LPS treatment. Te cemical compositions of HDL are sown in Table V. Tere were significant increases in plasma HDL pospolipid and free colesterol and a 44% decrease in HDL colesteryl ester concentration after LPS injection. Forty-eigt after injection te HDL colesteryl ester concentration was 67% lower tan base-line values, and tere was nearly a 3- fold increase in HDL triglyceride. HDL protein, pospolipid, and free colesterol were similar to preinjection concentrations. Te apoprotein profile of te density gradient regions was examined by SDS-polyacrylamide gradient gels. Aliquots of VLDL, ligt LDL, eavy LDL, and HDL were pooled separately from te gradients of four individual animals and analyzed (Fig. 2). Before LPS injection te major apoprotein of VLDL and LDL was apob and HDL (lune e) contained apoa-, A-11, and Cs. After LPS injection te major apoprotein of VLDL and ligt LDL (lunes f, g, j, and k) was still apob, but tere was an additional band at M, = 14,000 tat we ave previously identified as aposaa (15). Heavy LDL (lanes and i) contained very little apob and apoa- and apoe were te predominant apoproteins. Tis may represent contamination of eavy LDL wit HDL particles since LDL protein concentration decreased (Table 111) wile HDL protein concentration was uncanged (Table V). HDL fractions contained aposaa as well as apoa- after LPS injection (lanes i and m). Lipoprotein Morpology-Te canges in lipoprotein morpology associated wit endotoxin administration were examined by electron microscopy (Fig. 3). Prior to LPS administration, te VLDL were round in appearance and electronlucent wen negatively stained and viewed by electron microscopy (Fig. 3). After LPS treatment te VLDL were no longer uniformly round and some of te VLDL particles ad surface projections. Te LDL and HDL ad a typical round appearance before LPS administration, after wic, bot fractions contained many particles tat were discoidal in sape. Te HDL particles following LPS administration also tended to form rouleaux upon negative staining. HDL Size Heterogeneity-Tesize distribution of HDL subfractions was examined by gradient gel electroporesis in a subset of te animals. Peaks were classified as HDL2, HDL3,, or HDLX,,~ based on RF values of te HDL subfractions relative to bovine serum albumin according to te nomenclature of Blance et ul. (25). t as previously been sown tat te results of tis analysis correspond well wit results from density gradient centrifugation and analytical ultracentrifugation for monkey HDL subfractions (26). Peak areas were quantitated and are sown in Table V. Before LPS treatment alf of te HDL protein mass was distributed in HDL2, and te distribution did not cange significantly after LPS injection. HDL3. was initially 30% of te protein mass, but after LPS injection tis subfraction was dramatically reduced. Altoug tere were no detectable peaks in tis size range, tere was some protein mass wic could ave been HDL3, material or could ave been incomplete separation of HDL2 and HDLBb,c subfractions. Te percentage protein in HDL3b,c increased at after LPS injection, and tis was associated in a doubling of protein mass in tat subfraction (37 f 3 versus 70 f 6 mg/dl). Forty-eigt after injection te distribution of HDL subfraction protein was similar to base-line values. LCAT and Lipase Activities-Plasma LCAT activity was measured using an exogenous substrate. Te results are sown in Fig. 4 wic illustrates a precipitous drop in plasma LCAT

5 LPS-induced Lipoprotein Canges HDL LDL FG. 3. Electron micrograps of VLDL, LDL, and HDL isolated by density gradient centrifugation before (-LPS) and after (+LPS) injection of 300 pg of LPS/kg body weigt. VLDL - LPS + LPS TABLEV Effect of LPS injection on HDL subfraction protein distribution Percentage protein distribution was determined from HDL peak areas obtained from laser densitometer scans of 4-30% gradient gels. Plasma protein concentrations were determined by multiplying te % protein distribution by te total HDL protein concentration in plasma. Time nft.nr protein Plasma Protein 0 f16 (mg/du % 49.5b f f9.8 DSCUSSON 28.7 f C29 37 f3 70" f f4.2 ND' 40.1" 135 ND f f36 55 f10 37 f13 "HDL2, RF = ; HDL3., RF= ; HDLSb,c,RF = bmean 5 S.E. 'No detectable peaks. "p < 0.05, 0 uersus activity after LPS injection to 36% of base-line activity. LCAT activity was still 45% tat of preinjection values after but appeared to normalize by 72 after LPS injection in two animals followed for a longer periodof time. n five of te animals, plasma post-eparin lipase activity was measured before and after LPSinjection. Compared to preinjectionvalues post-eparin lipase activity was decreased 38% after LPS injection (39.6 & 4.0 versus.5 k 3.3 units/ml) HOURS AFTER LPS NJECTON FG.4. Wole plasma LCAT activity measured before and after LPS injection (300 pg/kg body wt) into eigt African green monkeys. LCAT activity was measured using an exogenous substrate asdescribed under "Materials and Metods." Eac symbol represents duplicate determinations for te timecourse of an individual animal. Tisstudy was initiatedtodeterminete effects of a relatively low dose of LPS on te plasma lipoproteins of African green monkeys. Tree-undred pg of LPS/kg body lipase weigt resulted ina decrease in LCAT and post-eparin activities and affected lipoprotein concentrations, compositions,anddistribution of te animals. Concentrations of plasma LDL and HDL decreased wile tose of VLDL increased. All lipoprotein fractions became enriced in triglyceride and depleted of colesteryl ester and, in te LDL and HDL fractions, te total amount of core material was reduced relative to surface, so tat many particles were discoidal in sape or sowed evidence of aving excess surface material. Many of te caracteristicsof tese plasma particles obtained after LPS injection were similar to toseof particles derived from recirculating perfusion of African green monkey livers in wic LCAT activity was very low (27, 30, 31) and from te plasma of LCAT-deficient uman beings (32, 33). Tus, tese findings suggest tat te lipoprotein particles found in plasma witin 2 days of LPS injection represent predominantly liver-derived particles tat areincompletely converted into mature particles. f tis is te case te LPS-treated African green monkey may serve as source of relatively unmodified epatic precursor particlesfor use for structural and metabolic studies. Of te lipoprotein compositional canges induced by LPS, te most striking was te decrease in colesteryl ester content. LCAT is considered te source of most plasma colesteryl ester in man but in colesterol-fed monkeys te liver is likely to make a significant contribution to te plasma colesteryl ester pool (30). Altoug we ave no data concerning te epatic contribution to te plasma colesteryl ester pool in tis study, we found a 55-64% reduction in LCAT activity, measured wit an exogenous substrate, concomitant wit a

6 reduction of plasma LDL and HDL colesteryl ester concentrations (Tables 11 and V). Sakaguci (5) as reported an increase in LCAT activity in mice treated wit LPS (7.7 mg/ kg), but an endogenous assay was utilized and it is unclear weter tis outcome was due to effects on enzyme activity or on substrate or a combination of bot. Tus, it appears likely tat te drop in plasma colesteryl ester content associated wit LPS treatment in tis study was caused by a decreased plasma LCAT activity. Altoug several studies ave demonstrated tat lipase activity is decreased by LPS in uiuo, to our knowledge tis is te first report to demonstrate a decrease in plasma LCAT activity wit a concomitant depletion of plasma colesteryl ester. To explain our observations, we ypotesize tat one or more monokines (i.e. interleukin-1, cacectin) released by macropages after interaction wit LPS inibits te syntesis of LCAT analogous to te situation wit lipoprotein lipase (12-14). n preliminary studies using te colesterol-fed cynomolgus monkey, we found tat bot plasma LCAT mass and activity were de- creased after intravenous injection of 100 pg of LPS.' Te decrease in plasma LCAT mass and activity also occurred wit injection of uman recombinant tumor necrosis factor (i.e. cacectin) but not wit uman recombinant interleukin- 1. Direct addition of LPS, tumor necrosis factor, or interleukin-1 to te LCAT assay of control plasma samples did not inibit te activity of LCAT. Tese data suggest tat LPS, tumor necrosis factor, and peraps oter monokines decrease LCAT activity by inibition of LCAT syntesis. Weter tese same compounds affect LCAT degradation as well must await furter studies. Te concentration of plasma VLDL was significantly greater at after LPS injection compared wit base-line concentrations and tis increase resulted from a 7-fold or greater concentration of all VLDL cemical constituents except colesteryl ester, wic was %fold iger. An increase in plasma VLDL after LPS injection as been documented in oter studies and is tougt to be secondary to te inibition of LPL activity (3,34-36). Available data support te concept tat macropages stimulated by an encounter wit LPS secrete several monokines including interleukin-1 and cacectin. Tese monokines ave been sown to inibit LPL activity in vitro and in uiuo (12-14). Tus, an inibition of LPL would lead to an accumulation of VLDL in plasma. Active LPL also appears to be important in te formation of LDL particles. Recently, Goldberg et al. (37) demonstrated tat inibition of LPL activity wit anti-lpl antiserum decreased te appearance of endogenously labeled apob into LDL of cynomolgus monkeys. Based on tese data te increase in plasma VLDL and decrease in LDL may ave resulted from te inibition of LPL wen LPS was given. Weter LPS also stimulates epatic secretion of VLDL as a result of increased plasma free fatty acid concentrations is unknown and could possibly contribute to te elevated concentrations of plasma VLDL. LPS treatment was associated wit a redistribution of HDL apoproteins. Before LPS treatment HDL particles contained predominantly apoa- wit smaller amounts of apoa-1 and C peptides (Fig. 2, lune e). After LPS treatment apoa- remained te major HDL apoprotein but te apoa-, A-11, and C peptides were replaced by a protein similar in size to tat of monkey aposaa (Fig. 2, lanes i and m), an acute pase reactant protein. Tis protein as a sequence identical to tat of uman aposaa in all but 8 of 103 amino acids.3 n a previous study using sort-term cair restraint of monkeys to induce aposaa, we concluded tat aposaa displaced apoa- * J. Parks, J. Albers, and W. Ettinger, unpublised observations. M. Lively, B. Auerbac, and J. Parks, unpublised observations. LPS-induced Lipoprotein Canges and apoa-1 from HDL particles (15). Since total HDL protein did not cange wit LPS treatment (Table V) and te relative amount of apoa-, A-11, and Cs decreased after LPS treatment (Fig. 2; lanes i and m) compared wit te 0 (lane e), it appears tat aposaa displaced HDL apoproteins in tis study also witout a net cange in total HDL protein. n our previous study tere was no evidence of a cange in te lipid composition or te total protein content of HDL (15), wile in te present study tere was a profound depletion of HDL colesteryl ester after LPS treatment. Tus, even toug te effects of LPS and cair restraint on HDL composition are distinct from one anoter, it appears in bot situations tat aposaa can displace apoa- and oter apoproteins from te surface of HDL. HDL particle size distribution, as monitored by protein distribution on gradient gels, was modified by LPS treatment even toug total HDL protein concentrations were constant (Table V). Since HDL protein concentration was uncanged wit LPS treatment, tis outcome may be te result of a redistribution of HDL lipid mass. HDLBb,= sized particles are te predominant species tat accumulate during recirculating perfusion of African green monkey livers (38). Tese HDL particles are predominantly discoidal in sape and are deficient in core colesteryl esters because of te low levels of LCAT in te perfusate (27). Upon incubation of te liver perfusate wit exogenous LCAT, HDL colesteryl ester content increased, te particles became round in appearance by electron microscopy and te size of te particles sifted to tat of HDL3, (38). Tus, a relative deficiency in LCAT activity caused by LPS injection may slow te conversion of HDL3b,c particles to HDL3, particles and result in te HDL subfraction protein distribution seen in Table V. However, at after LPS injection te distribution of HDL protein ad returned to base-line values even toug HDL particles remained discoidal in sape (Fig. 3), LCAT activity remained depressed (Fig. 4), and HDL particles were still deficient in colesteryl ester (Table V). t is possible tat by after LPS injection wen plasma triglycerides concentrations are elevated (Fig. l), te HDL3b,= particles become relatively enriced from te transfer of triglycerides from VLDL to HDL (Table V). Tis triglyceride enricment could result in a size increase of HDL3b,e particle so tat tey migrate in te HDL3, size range. Because our cemical compositions were measured on te entire spectrum of HDL particles, it is not possible to know weter te HDL3, particles were selectively enriced in triglycerides. t is clear from tis and oter studies tat experimental animals ave varying degrees of responsiveness to LPS. n our study monkeys given 300 pgof LPS/kg body weigt sowed dramatic canges in lipoprotein concentration, distri- bution, and composition wile mice given an equivalent of 1.7 mg/kg dose (assuming 30 g of body weigt) sowed no suc cange in HDL composition (39). Even correcting for differences in basal metabolic rate between te mouse (140 kcal/ kg/day; Ref. 40) and monkey ( kcal/kg/day), te animals in our study received approximately alf te amount of LPS given to mice. n recent years several studies ave used LPS or trauma to study acute pase reactant proteins wit regard to HDL metabolism (39, 41-45). Te results from tis study suggest tat in some experimental animals and at some doses tere may be marked perturbations of plasma lipoproteins tat accompany te appearance of acute pase reactant proteins. Tese perturbations, if not recognized, may complicate te interpretation of te study of acute pase reactant pro- teins and teir effect on lipoprotein metabolism. Te results of tis study as well as oters establis an

7 Yl LPS-induced Lii mprotein Canges interrelationsip between acute inflammation events and lipoprotein metabolism. ndeed, many patological states ave been associated wit one or more alterations in lipoprotein concentrations including increased plasma triglyceride concentrations and decreased total plasma and HDL colesterol (1-9). Te myriad of biological effects tat result from LPS are tougt to be mediated by monokines, but wit regard to te lipid canges only tumor necrosis factor as been sown to inibit LPL in vivo (12). To better understand te role of monokines on lipid metabolism during inflammatory events, more in vivo studies using te pure monokines will be necessary. Suc studies are currently underway in our laboratory using te nonuman primate model. Acknowledgments-We gratefully acknowledge te excellent assistance of Linda Odam in manuscript preparation. REFERENCES 1. Budd, D., and Ginsberg, H. (1986) Cancer 58, Coombes, E. J., Sakespeare, P. G., and Batstone, G. F. (1980) J. Trauma 20, Sakaguci, O., and Sakaguci, S. (1979) Microbiol. Zmmunol. 23, Fiser, R. H., Denniston, J. C., and Beisel, W. R. (1972) J. nfect. DU. 125, Sakaguci, S. (1982) Microbiol. Zmmunol. 26, Rouzer, C. A,, and Cerami, A. (1980) Mol. Biocem. Paristol. 2, Grossberg, S. E., and O Leary, W. M. (1965) Nature 208, Farstci, D., and Lewis, V. J. (1968) J. Bacteriol. 95, Oster, P., Mucowski, H., Heuck, C. C., and Sclierf, G. (1981) Klin. Wocenscr. 59, Morrison, D. C., and Ulevitc, R. J. (1978) Am. J. Patol. 93, Elin, R. J., and Wolff, S. M. (1976) Annu. Reu. Med. 27, Semb, H., Peterson, J., Tavernier, J., and Olivecrona, T. (1987) J. Biol. Cem. 262, Price, S. R., Mizel, S. B., and Pekala, P. H. (1986) Biocem. Biopys. Acta 889, Beutler, B., Maoney, J., Le Trang, N., Pekala, P., and Cerami, A. (1985) J. Exp. Med. 161, Parks, J. S., and Rudel, L. L. (1985) J. Lipid Res. 26, Rudel, L. L., Parks, J. S., and Carroll, R. M. (1983) in Dietary Fats and Healt (Perkins, E. G., and Visek, W. J., eds) pp , American Oil Cemists Society, Campaign, L 17. Rudel, L. L. (1980) in Proceedings of te First Annual Symposium on Te Use of Nonuman Primates in Cardiouusculnr Researc (Kalter, S. S., ed) pp , University of Texas Press, San Antonio. TX 18. Rudel, L. L., Reynolds, J. A,, and Bullock, B. C. (1981) J. Lipid Res. 22, Warnick, G. R., and Albers, J. J. (1978) J. Lipid Res. 19, Rudel, L. L., and Morris, M. D. (1973) J. Lipid Res. 14, Lowry, 0. H., Rosebroug, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Cem. 193, Fiske, C. H., and SubbaRow, Y. (1925) J. Biol. Cem. 66, Sardesai, V. M., and Manning, J. A. (1968) Clin. Cem. 14, Radin, N. S. (1969) Metods Enzymol. 14, Blance, P. J., Gong, E. L., Forte, T. M., and Nicols, A.V. (1981) Biocem. Biopys. Acta 665, Babiak, J., Lindgren, F. T., and Rudel, L. L. (1988) Arteriosclerosis 8, Jonson, F. L., Babiak, J., and Rudel, L. L. (1986) J. Lipid Res. 27, Cen, C., and Albers, J. J. (1982) J. Lipid Res. 23, Winer, B. J. (1971) Statistical Principles in Experimental Design, McGraw-Hill, New York 30. Jonson, F. L., St. Clair, R. W., and Rudel, L. L. (1985) J. Lipid Res. 26, Jonson, F. L., St. Clair, R. W., and Rudel, L. L. (1983) J. Clin. Znuest. 72, Glomset, J. A., Norum, K. R., and King, W. (1970) J. Clin. Znuest. 49, Norum, K. R., Glomset, J. A,, Nicols, A. V., and Forte, T. (1971) J. Clin. nuest. 50, Bagby, G. J., and Spitzer, J. A. (1980) Am. J. Pysiol. 238, H325- H Scoll. R. A.. Lane. C. H.. and Bazbv. - -, G. J. (1984) J. Sure. - Res. 37, Kawakami, M., and Cerami, A. (1981) J. Exp. Med. 154, Goldberg,. J., Le, N., Ginsberg, H. N., Krauss, R.M., and Lindgren, F. T. (1988) J. Clin. Znuest. 81, Baiak, J., Tamaci, H., Jonson, F. L., Parks, J. S., and Rudel, L. L. (1986) J. Lipid Res. 27, Hoffman, J. S., and Benditt, E. P. (1982) J. Biol. Cem. 257, Spector, W. S. (ed) (1956) in Handbook of Biological Data p. 258, W. B. Saunders Co., Piladelpia, PA 41. Skogen, B., Borresen, A. L., Natvig, J. B., Berg, K., and Micaelsen, T. E. (1979) Scand. J. Zmmunol. 10, Hoffman, J. S., and Benditt, E. P. (1983) J. Clin. Znuest. 71, Coetzee, G. A., Stracan, A. F., van der Westuyzen, D. R., Hoppe, H. C., Jeena, M. S., and de Beer, F. C. (1986) J. Biol. Cem. 261, Moon, E. A., Mackinnon, A. M., and Barter, P. J. (1984) Biocim. Biopys. Acta 796, Munford, R. S., Andersen, J. M., and Dietscy, J. M. (1981) J. Clin. Znuest. 68, Parks, J. S., and Rudel, L. L. (1979) J. Biol. Cem. 254,