papers on methodology

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1 ppers on methodology A low temperture flottion method to rpidly isolte lipoproteins from plsm H. Tong, H. R. Knpp, nd M. VnRollins 1 Division of Clinicl Phrmcology, Deprtment of Internl Medicine, College of Medicine, The University of Iow, Iow City, IA Abstrct To minimize oxidtive modifiction, low temperture, sequentil flottion method ws developed to isolte plsm lipoproteins in 18 h using benchtop ultrcentrifuge. The protein distributions were chrcterized using grose nd SDS-polycrylmide gel electrophoresis, nd n SDS-Lowry protein ssy. The lipid distributions were ssessed using gs chromtogrphy mss spectrometric ssy for cholesterol nd n enzymtic ssy for triglycerides. To vlidte the rpid flottion method, lipoproteins were lso isolted from the sme plsm smples using modified Hvel et l. flottion method ( J. Clin. Invest. 34: , 1955). The sme lipoproteins nd polipoproteins were present in frctions of comprble density, nd the summed recoveries of protein, cholesterol, nd triglyceride were lso identicl for the Hvel et l. nd rpid flottion procedures. Likewise, the mount of cholesterol nd triglyceride in corresponding very low, intermedite, nd low density lipoprotein (VLDL/IDL nd LDL) frctions ws the sme for the two flottion procedures. The triglyceride nd cholesterol levels in high density lipoprotein (HDL) isolted by rpid flottions, however, were 9 12% higher thn in the HDL s isolted by Hvel et l. Becuse 9 12% increse in the HDL frction reflects only 1 4% of the totl triglyceride nd cholesterol in plsm, we conclude tht, while mintined t 4 C, lipoproteins were quntittively isolted from humn plsm in 1 dy. Tong, H., H. R. Knpp, nd M. VnRollins. A low temperture flottion method to rpidly isolte lipoproteins from plsm. J. Lipid. Res : Supplementry key words ultrcentrifugtion humn plsm very low density lipoproteins low density lipoproteins high density lipoproteins mss spectrometry cholesterol tricylglycerides utooxidtion employed by Hvel, Eder, nd Brgdon (1) to resolve lipoproteins into four clsses: VLDL (d g/ml); IDL (1.006 d 1.019); LDL (1.019 d 1.063); nd HDL (1.063 d 1.21). The Hvel et l. flottion method is frequently used to isolte lrge mounts of lipoproteins for the purpose of biologicl testings, e.g., the genertion of oxidized LDL. Unfortuntely, the method requires severl dys to be completed. Studies hve shown tht polipoproteins redistribute mong different clsses of lipoproteins during prolonged centrifugtions; moreover, the integrity of the lipoprotein prticles my be compromised becuse lbile polipoproteins locted on lipoprotein surfces cn be preferentilly degrded (2 4). The presence of free rdicls in plsm (5) lso rises the possibility tht lipid peroxidtion nd utoxidtion will occur t the reltively high temperture (20 C) trditionlly used (6). When lipophilic nti-oxidnts nd free rdicl scvengers re prophylcticlly dded to plsm, they cn become sequestered by the plsm lipids, nd render the lipoproteins indequte for oxidtion studies. Yet, without nti-oxidnts, the use of prolonged centrifugtions t 20 C fvors the oxidtion of ntive lipoproteins. Common benchtop ultrcentrifuges cpble of generting very high g forces, hve recently been used to isolte lipoproteins for both preprtive nd nlyticl purposes. To minimize the processing rtifcts described bove, Brousseu et l. developed sequentil flottion method for the preprtive isoltion of lipoproteins. By use of Beckmn TL 100 benchtop ultrcentrifuge (up to 100,000 rpm) nd fixed-ngle rotor, they isolted serum lipoproteins t 15 C in 9.5 cumultive cen- We re interested in estblishing rpid preprtive method to isolte lipoproteins in mnner tht minimizes their oxidtive modifiction. Ultrcentrifugtion methods hve commonly been used to preprtively isolte lipoproteins from plsm or serum. One of the most used ultrcentrifugtion techniques involves sequentil flottions in medi of incresing densities, nd ws first Abbrevitions: VLDL, very-low density lipoproteins; IDL, intermedite density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; EDTA, ethylenediminetetrcetic cid; SDS, sodium dodecyl sulfte; PAGE, polycrylmide gel electrophoresis; %T, totl crylmide content (w/v); %C bis, the rtio of bis-crylmide to crylmide monomer (w/w); GC/MS, gs chromtogrphy mss spectrometry; RSD, reltive stndrd devition. 1 To whom correspondence should be ddressed Journl of Lipid Reserch Volume 39, 1998

2 trifugtion hours. However, their preprtive method did not test the use of low tempertures; moreover, second centrifugtion step ws required to minimize lbumin contmintion. Recently, flottions with benchtop centrifuges hve lso been combined with precipittion techniques to isolte lipoproteins for nlyticl purposes. For exmple, Leonhrdt et l. (8) described procedure to flot VLDL using benchtop ultrcentrifuge t 17 C, nd used precipittions to seprte LDL from HDL. The precipittion step eliminted the prolonged centrifugtion times required for floting HDL. Similrly, Cthcrt nd Dominiczk (9) reported isoltion of VLDL in 2.5 h t 4 C, nd seprted HDL from LDL by precipitting HDL. Fletcher, Brnes, nd Frish (10) lso reported rpid semi-micro procedure for seprting VLDL, IDL, nd LDL t 20 C using benchtop ultrcentrifuge fter HDL precipittion. The precipitted lipoproteins re commonly contminted with plsm proteins; moreover, their lipid composition my differ somewht from tht of nlogous lipoproteins obtined by flottion techniques (11). Finlly, before precipitted lipoproteins cn be used for biologicl studies, they must first be resolubilized so tht precipitting gents my be completely removed by dilysis. For these resons s well s their cpcity to be redily utomted, the hybrid flottion precipittion methods re generlly used for clinicl nlyses rther thn for preprtive purposes. To dte, no low temperture sequentil flottion method using benchtop ultrcentrifuge to isolte lipoproteins for biologicl studies hs been described. Mintining lipoproteins t low temperture during processing will minimize their ex vivo oxidtion. Currently, there is n intense effort to identify the oxidtion products of phospholipids, sterols nd polipoproteins tht re formed in vivo nd promote therosclerosis. Oxidized lipids such s lipid peroxides (12 14), oxysterols (15, 16) nd polipoproteins modified with ldehyde dducts (17, 18) exhibit biologicl ctivities tht re of potentil physiologicl, pthologicl or phrmcologicl importnce (16, 19). Moreover, mny studies hve indicted the existence of lipid epoxides nd oxysterols in plsm nd bile, though their origins re uncler (5, 20 23). However, estblishing the in vivo concentrtions of lipoprotein epoxides nd oxysterols hs been rther difficult, becuse LDL is unstble nd highly prone to oxidtion during processing nd storge (24). As the in vivo concentrtions of lipid epoxides nd oxysterols in plsm re likely to be very low, rtifcts cused by oxidtion during smple processing could present serious problem for the interprettion of such studies. In the present report, we describe sequentil flottion method to isolte lipoproteins t 4 C from 1.0 ml of plsm in 18 h. The VLDL/IDL, LDL nd HDL frctions were lmost freed of lbumin contmintion by optimizing both the centrifugtion times nd the sites for tube trnsections. To vlidte the method, the contents of the isolted frctions were compred to those generted by the Hvel et l. method (1). MATERIALS AND METHODS Isoltion of humn plsm Six helthy volunteer dults (4 mle nd 2 femles) were fsted for t lest 12 h before hving 100 ml of blood withdrwn from their ntecubitl veins. The blood ws collected into two 60- ml syringes contining 0.8 ml of nticogulnt (0.2 m ethylenediminetetrcetic cid (EDTA) in 150 mm NCl, ph 7.4). After being divided eqully between two plstic tubes, the 50-ml venous smples were gently inverted in the presence of 2.0 ml 150 mm NCl tht contined nti-bcteril gents (5 g sodium zide, 4 mg chloromphenicol, nd 4.0 mg gentmicin sulfte) nd the kllikrein inctivtor protinin (200 units); the mixture ws prepred fresh dily (25). Upon centrifuging the smples t 4000 g mx nd 4 C for 30 min, the plsm ws removed using plstic pipettes, mixed with benzmidine (proteolysis inhibitor) nd phenylmethylsulfonyfluoride (serine proteses inhibitor), ech to finl concentrtion of 1.0 mm, nd trnsferred to Quick-Sel centrifuge tubes (Beckmn, Plo Alto, CA) for the sequentil flottion procedure. In the following studies, ll the required centifugtion equipment nd ccessories were purchsed from Beckmn. Isoltion of lipoproteins by the Rpid Flottion procedure Lipoprotein frctions were isolted from four 1.0-ml liquots of plsm using benchtop ultrcentrifuge (Optim TLX) nd fixed-ngle rotor (TLA 120.2) operting t 120,000 rpm (627,000 g mx ) nd mintined t 4 C. The lipoproteins were seprted in 2.0 ml dome-topped Quick-Sel tubes (Polyllomer, mm) which hve smple cpcity twice tht of similrly sized open tubes. Approprite centrifugtion times for lipoproteins t 20 C were clculted using the eqution k 1 /t 1 k 2 /t 2 (k, fctor; t, spin time), nd compenstion of 12% to LDL nd 9% to HDL ws dded for every 5 degree decrese in temperture (11). The incresed centrifugtion time ws necessry to compenste for n incresed solution viscosity tht occurs t 4 C. Assuming 12% compenstion for VLDL/IDL, the pproprite times were clculted to be 4.93 h for VLDL/IDL, 4.93 h for LDL, nd 9.04 h for HDL isoltion t 4 C. In close greement with the k fctor nd low temperture considertions, experimentlly the VLDL/ IDL ws completely nd reproducibly seprted in 4 h, LDL in 5 h, nd HDL in 9 h (vide infr). Isoltion of VLDL/IDL. One ml plsm nd ml of NBr (38% w/v) were dded with Hmilton syringes to Quick-Sel tube. The volume nd density of ech plsm smple were djusted to 2.0 ml nd g/ml, respectively, t 20 C using 0.15 m NCl contining 1.0 mm EDTA, nd digitl blnce. Ech tube ws het-seled (Quick-Sel Tube Seler), centrifuged for 4.0 h t 627,000 g mx nd 4 C, nd trnsected mm (10.5 setting) from the bse using Teflon-coted bldes nd grduted slicer (CentriTube Slicer). Reproducible trnsections were criticl becuse the cler spce tht intervened between floted nd nonfloted lipoproteins is much less thn in the long centrifuge tubes used in the Hvel et l. procedure. About 0.8 ml ws present in the upper section; the floted VLDL/IDL contents were collected nd combined with sline wshes of the upper section. Isoltion of LDL. The remining solution in the lower section ws trnsferred to new Quick-Sel tube. The finl volume nd density of the smple were djusted to 2.0 ml nd g/ml, respectively, t 20 C using 38% NBr, 0.15 m NCl, nd digitl blnce. Upon being centrifuged for 5.0 h t 627,000 g mx nd 4 C, ech tube ws trnsected 17.5 mm mm (11.0 setting) from its bse. About 0.75 ml ws present in the upper section, nd its floted LDL contents were collected nd combined with sline wshes of the upper section. Tong, Knpp, nd VnRollins Isoltion of lipoproteins by sequentil flottions t 4 C 1697

3 Isoltion of HDL. Designted s the cler zone beneth the LDL, the topmost 0.1 ml in the bottom section ws discrded to permit density djustments. The remining 1.15 ml in the bottom section ws trnsferred to new Quick-Sel tube, nd its volume nd density were djusted to 2.0 ml nd 1.21 g/ml, respectively, t 20 C using 38% NBr nd digitl blnce. Ech tube ws centrifuged for 9.0 h t 627,000 g mx nd 4 C. Upon trnsecting the tube 16.0 mm (10.0 setting) from the bse, bout 0.9 ml ws isolted in the upper section, nd its floted HDL content ws collected nd dded to sline wshes of the upper section. Isoltion of lipoproteins by the Hvel et l. flottion procedure Isoltion of VLDL/IDL. Thirty-five ml plsm nd ml of 38% NBr were trnsferred to dome-topped Quick-Sel tube (Polyllomer, mm). The volume nd density of ech plsm smple were djusted to 39 ml nd g/ml, respectively, using digitl blnce nd 0.15 m NCl contining 1.0 mm EDTA. Upon being seled, ech tube ws trnsferred to fixedngle rotor (Type 60), nd centrifuged (L7-55) for 20 h t 50,000 rpm (176,200 g mx ) nd 20 C. Ech tube ws trnsected 58 mm from its bse by use of stinless steel blde; bout 9.5 ml of the floted VLDL/IDL ws collected from the top section. The upper surfce of the blde nd inside surfce of the excised upper section were both wshed three times with 0.15 m NCl; the wshes were dded to the isolted VLDL/IDL frction. Isoltion of LDL. The remining smple present in the lower section ws divided eqully between two new Quick-Sel tubes. The volume nd solution density in ech tube were djusted to 39 ml nd g/ml, respectively, using 38% NBr, 0.15 m NCl, nd digitl blnce. Upon being seled, ech tube ws centrifuged for 20 h t 176,200 g mx nd 20 C, nd trnsected 51 mm from its bse. About 19 ml ws isolted in the upper tube section, collected, nd combined with sline wshes of the blde nd upper tube section. Isoltion of HDL. Designted s the cler zone beneth the LDL, the topmost 10 ml in the bottom sections ws discrded to permit density djustments. The remining infrntnt in the two bottom sections ws trnsferred to two new Quick-Sel tubes. The volume nd density of ech smple were incresed to 39 ml nd 1.21 g/ml, respectively, with 38% NBr nd 0.15 m NCl, nd the tubes were seled nd centrifuged for 40 h t 176,200 g mx nd 20 C. After ech tube ws trnsected 51 mm from its bse, the HDL ws isolted in 19 ml, nd combined with sline wshes from the blde nd upper tube section. Designted s the cler zone beneth the HDL, the topmost 10 ml in the bottom tube section ws lso collected to determine whether ny cholesterol ws indvertently left behind. Chrcteriztion of lipoprotein frctions Agrose gel electrophoresis ws used to identify the lipoprotein constituents of floted frctions. One ml of ech VLDL/IDL, LDL, nd HDL frction ws deslted nd concentrted to bout 30 l by filtrtion (Centricon 50, Amicon, Beverly, MA) t 5000 g, 4 C for 60 min. The mount of protein or lipid lost during filtrtion from retined lipoproteins ws ssumed to be the sme for comprble frctions. About l of ech concentrted smple ws loded with modified Hmilton syringe onto pre-cst 1% grose gel film (Universl Gel 8, Cib-Corning). Humn lipoprotein stndrds, generously provided by Dr. Dvid Chppell, U. of Iow, were similrly pplied for identifiction purposes. At room temperture nd under constnt 90 V field, the lipoproteins were seprted in 35 min using 50 mm brbitl buffer (ph 8.6; Universl PHAB buffer, Cib-Corning). Upon being blotted, the gel ws dried t 60 C for bout 1 h, nd stined for 30 min t room temperture with Ft Red 7B (2.25 mg dissolved in 10 ml methnol nd 2 ml 0.1 N NOH). The gel ws destined in 70% methnol nd dried overnight t room temperture. Grdient gel electrophoresis ws used to identify polipoprotein components of the floted frctions. Verticl slb-gels (0.75 mm 11 cm 14 cm) were prepred contining 5 20% polycrylmide grdient (26). In brief, 7.0 ml of 5% T, 0.8% C bis ws mixed with 7 ml of 20% T, 0.8% C bis using grdient mker (SG 50, Hoeffer Scientific, Sn Frncisco, CA), nd pumped t 7 ml/min into sndwich chmber (Hoefer Scientific). Both solutions were previously fortified with 0.4% SDS plus 1.5 m Tris (ph 8.8), nd hd been degssed under house vcuum for t lest 30 min. Complete polymeriztion occurred within 30 min fter dding 50 l of 10% mmonium persulfte nd 5 l N,N,N,N-tetrmethylene-ethylenedimine. A stcking gel (0.75 mm 2 cm 14 cm) ws similrly generted in 1 2 h using 4% T, 0.8% C bis. On the sme or next dy, smples from whole plsm nd lipoprotein frctions were resolved using n electrophoresis chmber (SE 600, Hoefer) mintined t 15 C by circulting cooler. Stndrds (SDS PAGE Moleculr Weight Stndrds-High; Bio- Rd Lbortories, Richmond, CA) were pplied to the outermost lnes to id polipoprotein identifictions. All protein-contining smples (c. 10 g) were initilly heted t 60 C for 1 h with 0.25 vol of 60 mm Tris-HCl (ph 6.8), 25% glycerol, 2% SDS, 14.4 mm 2-mercptoethnol, nd 0.1% bromophenol blue. Upon being diluted with deionized wter to 50 l, the dentured proteins were subjected to electrophoresis t constnt 60 ma for 4 5 h. The gel ws trnsferred to 0.1% Coomssie blue R-250 solution for overnight stining. Excess stin ws removed by rinses with methnol cetic cid wter 40:10:50 (v/v/v). An SDS-Lowry protein ssy ws used to mesure protein concentrtions in ech of the floted frctions (not previously de-slted by filtrtion). Preliminry results indicted tht the NBr concentrtions used in the present study hd no effect on protein determintions, nd confirmed previous findings (27). After solubliztion with SDS nd rection with Folin-Cioclteu phenol regent, the protein content of ech lipoprotein frction ws determined spectrophotometriclly s equivlents of bovine serum lbumin (Pierce Chemicl Co) (28). The men reltive stndrd devition (RSD; n 10) of ten lbumin concentrtions (10 60 g) ws 2.83%. An isotope-dilution ssy for cholesterol using gs chromtogrphy mss spectrometry (GC/MS) ws used to ssess lipoprotein recoveries. Cholesteryl esters were sponified using low temperture modifiction of the Ishikw micromethod (29). In brief, 10 l of plsm or floted lipoprotein ws dded to 1.0 ml Rectivil (Pierce Chemicl Co., Rockford, IL) contining 10 g [26,26,26,27,27, 27-2 H 6 ]cholesterol (98.4% isotopic purity; Medicl Isotopes, Pelhm, NH). Tetrmethylmmonium hydroxide (6.25 mg in 100 l of 2-propnol methnol 3:1) ws dded, nd the vils were flushed with rgon for 30 sec, seled with Teflon-lined cps, nd periodiclly mixed t 4 C over 6-h period. The sponified lipids were extrcted by mixing with tetrchloroethylene (50 l) nd wter (200 l) for 1.0 min. After 10-min centrifugtion t 1250 g mx nd 4 C, the lower orgnic phse contining unesterified cholesterol ws collected nd trnsferred to 100- l Rectivil. Prior to quntittion by GC/MS, the unesterified cholesterol ws converted to trimethylsilylether derivtive. In brief, the sponified smples were dried under nitrogen strem, nd suspended in 50 l of N,O-bis(trimethylsilyl)triflorocetmide (Pierce Chemicl Co.) nd 50 l cetonitrile. Upon being flushed with rgon for 30 sec, ech vil ws cpped, shken, nd left overnight t room temperture. The next morning, the smple ws dried under strem of nitrogen nd dissolved in 100 l decne. The trimethylsilyether of cholesterol ws nlyzed using cpillry gs chromtogrphy (5890, Hewlett-Pckrd). About one 1698 Journl of Lipid Reserch Volume 39, 1998

4 hundredth of the totl extrct ws deposited vi duck-bill injector (Hewlett-Pckrd) into fused silic cpillry column (0.25 mm (i.d.) 30 m) contining 0.25 m film of 5% diphenyl 95%dimethylpolysiloxne (DB-5-HT, J&W, Rncho Cordov, CA). The liner velocity of the helium crrier gs ws progrmmed to remin constnt t 25 cm/sec. One min fter injection, the oven temperture ws incresed 70 C/min from 170 C to finl constnt temperture of 275 C. The injector temperture ws progrmmed to be 3 C bove tht of the oven, while the interfce remined t 280 C. Under these conditions, the retention time for cholesterol ws min. Quntittion ws performed using n isotope dilution technique nd qudrupole mss spectometer (5970B, Hewlett-Pckrd). Deuterted cholesterol ws used s the internl stndrd to normlize for vribility in recoveries during smple processing. To ssess reltive reponses, vrying mounts of d 0 -cholesterol ( ng) were weighed nd mixed with 100 ng of d 6 -cholesterol. The d 0 /d 6 mixtures were silylted (see bove), nd constnt mounts of d 6 -cholesterol (1.0 ng/1.0 l) were injected into the GC/MS system. After the protium nd deuterted cholesterols were ionized t 70 ev, the currents generted by m/z 458/464 (M) nd 368/374 [M (CH 3 ) 3 SiOH] were integrted. Using regression nlysis, the d 0 /d 6 rtios were found to be highly liner (r ), i.e., for weight rtios vrying between 1:10 to 5:1, the re rtio of m/z 458/464 ws (weight rtio of cholesterol/[d 6 ]cholesterol) Moreover, injection of the sme smple ten times yielded RSD of 0.64% for 500 mg/dl nd 1.45% for 4.5 mg/dl cholesterol. Such low intrssy vribility is criticl for detecting smll differences in cholesterol recoveries. Moreover, whole plsm cholesterol concentrtions (mg/dl) determined by GC/MS (178, 244, 123, 168, 171, nd 151) greed within 3.3% of those determined enzymticlly (181, 251, 126, 172, 174, nd 156, respectively) (30). Thus, the preliminry studies indicted tht the GC/MS ssy hd 1.5% precision nd 3.3% ccurcy. An enzymtic ssy ws used to quntitte the triglyceride content of floted lipoproteins. Triglycerides were quntitted using glycerol-3-phosphte oxidse method (31) modified for use with microtiter plte reder (THERMO mx, Moleculr Devices Corp., Sunnyvle, CA). In brief, 2 15 l of lipoprotein frctions ws diluted to 30 l with 0.1 m sodium phosphte (ph 7.6) nd mixed with 100 l of solution A contining glycerol kinse (EC ), denosine triphosphte, glycerol-3-phosphte oxidse ( ), peroxidse (EC ), nd 4-chlorophenol (Boehringer Mnnhein, GmbH). After 5-min incubtion t 25 C to remove free glycerol, the mixture ws shken for 1 min with 100 l solution B contining triglyceride lipse (EC ) plus 4- minontipyrine (Boehringer Mnnheim), nd incubted 10 min t 25 C. Over rnge of mg/dl glycerol, liner mounts of the chromogen 4-(p-benzoquinonemonoimino) phenzone were generted (r % RSD; n 6) s monitored by bsorption t 490 nm ginst regent blnk. On the sme dy, replicte ssys of lipoprotein frctions contining 109, 122, nd 307 mg glycerol/dl vried by % RSD (n 10 12). Sttistics. The contents from corresponding frctions were nlyzed s rtios, nd evluted using one smple t-test. A vlue of P 0.05 ws considered to be sttisticlly significnt. RESULTS Use of high-speed, benchtop ultrcentrifuge permitted isoltion t 4 C of three lipoprotein frctions in 24 h Fig. 1. Times nd tempertures of Hvel et l. nd rpid flottion procedures. Abbrevitions: VLDL, very low density lipoproteins; IDL, intermedite density lipoproteins; LDL, low density lipoproteins; nd HDL, high density lipoproteins. (Fig. 1). Enzymtic trnsformtions nd utooxidtions of the lipid constituents should be minimized by the shorter centrifugtions t 4 C s compred to the procedure used by Hvel et l. (1). Becuse LDL nd HDL frctions must first be deslted before their lipids cn be quntittively extrcted by the Folch or Bligh-Dyer methods, it tkes up to 24 h before ll enzymtic ctivity is eliminted by extrctive isoltion. In contrst, similr extrctions cnnot be performed for up to 5 dys when lipoproteins re isolted by the Hvel et l. procedure (Fig. 1). To determine whether the rpid flottions generted frctions with contents comprble to those of the Hvel et l. procedure, six plsm smples were subjected to both flottion procedures. The contents of the resulting frctions were nlyzed using electrophoretic techniques, nd lipoprotein recoveries were ssessed by protein, cholesterol, nd triglyceride mesurements. In ddition, the summed recovery ws compred to whole plsm vlues. The types of lipoproteins present in the three frctions were identified by grose gel electrophoresis; Fig. 2 illustrtes typicl results from one of the six plsm smples. Neither of the two LDL frctions contined pprecible HDL when lipoproteins were identified by co-migrtions with humn stndrds; however, becuse of pronounced tiling of the VLDL/IDL frction (Fig. 2), it ws uncertin s to whether significnt mounts of VLDL/IDL occurred in the LDL frction. The types of proteins present in ech frction were exmined using SDS polycrylmide gel electrophoresis; Fig. 3 illustrtes typicl results from the plsm smples. Intermedite-sized polipoproteins nd lbumin were identified from semi-logrithmic plots of moleculr weight ver- Tong, Knpp, nd VnRollins Isoltion of lipoproteins by sequentil flottions t 4 C 1699

5 Fig. 2. Agrose gel electrophoresis of lipoprotein frctions isolted by the Hvel et l. nd rpid flottion methods. Frctions contining lipoproteins in n mount equivlent to tht present in l of whole plsm were spotted in prllel lnes onto pre-cst grose film. The two HDL frctions were pplied to lnes 1 nd 2 (strting from the left side); the two LDL frctions to lnes 3 nd 4; the VLDL/IDL frctions to lnes 5 nd 6; nd the totl lipoprotein frctions (density 1.21 g/ml) were pplied to lnes 7 nd 8. Individul lipoproteins were resolved using constnt voltge, nd visulized using Ft Red 7B stin. Abbrevitions: H, Hvel et l.; R, rpid flottions. sus R f. ApoB-100 (M r 200 kd) ws the mjor polipoprotein in the VLDL/IDL nd LDL frctions. Becuse no poa-i (M r 23 kd, vs. expected 28 kd) ws evident in the LDL frctions, the polycrylmide gel electrophoresis studies confirmed tht the HDL frction ws essentilly free of LDL. Moreover, polipoproteins E-1, E-2, nd E-3 (M r 34.9, 33.3 kd vs. expected 34 kd) nd poc-ii nd poc-iii (M r 14.4 kd) were primrily in the VLDL/IDL frctions. Thus, in contrst to the grose gel results, polycrylmide gel electrophoresis dt indicted tht there ws little contmintion of the LDL frction by dense IDL. Therefore, both electrophoresis studies indicted tht similr types of lipoproteins hd been isolted by the two flottion procedures. The quntity of protein in comprble frctions ws very similr (Tble 1). Although lbumin (observed M r 69 kd, vs. expected 66 kd) ws vribly present in the three frctions (Fig. 3), its contributions were miniml in the Rpid Flottion procedure. Serum lbumin is considered to be the predominnt protein contminnt of lipoproteins isolted by ultrcentrifugtion methods (7, 32). After repetedly experiencing difficulty in removing lbumin from the HDL frction by the Hvel et l. procedure, we divided the VLDL/IDL nd LDL infrntnts between two tubes before subjecting them to prolonged centrifugtions. Using this dilution technique, we obtined the highest qulity lipoproteins from Hvel s method for reference purposes. As judged by grose gel electrophoresis nd SDS 5 20% PAGE, lipoprotein frctions isolted using our rpid method were of purity equl to those obtined by the slightly modified Hvel et l. method. Under these conditions, the totl protein recovered from 1.0 ml plsm by the Hvel et l. procedure ws pproximtely 2.1 mg/ml. Very close to the sme mount of totl protein ws recovered by the rpid flottion method; replicte determintions hd 3% vribility (Methods section). Likewise, the mount of protein ws virtully the sme in comprble subfrctions. Thus, both flottion methods produced frctions with mtching mounts of polipoproteins nd protein contminnts. To precisely ssess lipoprotein recoveries, the mount of totl cholesterol in ech frction ws mesured using GC/MS methods. However, smll mounts of cholesterol were unvoidbly lost prior to these nlyses. After ech LDL frction ws collected, volume ws discrded from the top of the remining solution, the so-clled LDL cler zone. Without removing this mount of solution, the totl Fig. 3. SDS-PAGE (5 20%) grdient gel electrophoresis of dentured lipoproteins isolted by the Hvel et l. nd rpid flottion methods. Stndrds with moleculr weights rnging from 14,400 to 200,000 were pplied to the outermost lnes of 5 20% SDS-PAGE. Excepting the totl lipoprotein frctions (density 1.21 g/ml), bout 10 g protein from the frctions isolted by Hvel et l. nd rpid flottion methods ws pplied in the remining lnes: HDL to lnes 2 nd 3; LDL to lnes 4 nd 5; nd VLDL/IDL to lnes 6 nd 7. In ddition to the mjor polipoprotein B 2 (M r 200 kd), the smll polipoproteins E 1 3 (M r 34 kd) nd C 2 3 (M r 14.4 kd were lso evident on the 5 20% SDS-PAGE grdient (lnes 6 nd 7). To lnes 8 nd 9, more thn 10 g protein of the totl lipoprotein frctions ws pplied in order to permit redy visuliztion of the minor polipoproteins. The mjor nd minor polipoproteins were resolved by pplying constnt current, nd visulized by stining with Coomssie blue. Abbrevitions: H, Hvel et l.; R, rpid flottions Journl of Lipid Reserch Volume 39, 1998

6 TABLE 1. Protein content of frctions isolted by Hvel et l. (1) nd rpid flottion procedures Lipoprotein Frction Hvel et l. Flottion Procedure Rpid Flottion Procedure b VLDL/IDL % LDL % HDL % Summed protein in ll frctions % Dt represent men mg SD (n 6) of protein isolted from 1.0 ml of plsm. The protein contents were directly ssyed without concentrting or deslting the isolted frctions. b To more redily show differences from the reference method, the dt re presented s men percent SEM (n 6) of the protein vlue in the corresponding Hvel et l. frction. volume of the smple fter the density djustment would be lrger thn the volume of centrifuge tube. The mount of cholesterol lost to the LDL cler zone represented 2.1% 1.1 nd 0.4% 0.2 of the plsm cholesterol level (161 mg/dl 37, n 6), respectively, in the Hvel et l. procedure (1) nd the current rpid flottion procedure, respectively. An dditionl 0.7% 0.7% of cholesterol ws lost to the HDL cler zone in the Hvel et l. procedure; however, no cholesterol ws detectble in the HDL cler zone in the rpid flottion procedure. In summry, % of the totl plsm cholesterol ws discrded before the cholesterol content of individul lipoprotein frctions could be nlyzed. Cholesterol recoveries for the two procedures represented 96% of the totl mount vilble in whole plsm (Tble 2). Becuse the experimentl error for cholesterol determintions ws less thn 1.5% (Methods section), these results indicted tht the cholesterol recoveries were indeed less thn 100%. However, s 0.4% nd 2.8% of the totl cholesterol hd been discrded with the cler zones, both procedures ccounted for % of the lipoprotein cholesterol originlly present in plsm. Anlysis of the cholesterol distributions reveled tht comprble frctions hd similr cholesterol contents. Both flottion procedures yielded similr mounts of cholesterol in the VLDL/IDL frction, s well s in the LDL frction (Tble 2). With such vribility in the six plsm smples, only differences of 12% (VLDL/IDL) nd 6% (LDL) would hve been detected with power of Surprisingly, the HDL frction isolted by the rpid flottion procedure hd 12% more cholesterol thn the sme frction isolted by the Hvel et l. procedure. However, becuse the dditionl cholesterol represented only 3.4% (12.2% 28.6) of the plsm totl cholesterol, the totl recovery nd distribution of cholesterol were very similr for the two flottion methods. The rpid flottion procedure yielded similr totl mounts of triglyceride s the Hvel et l. procedure (Tble 3). Becuse the recovery in the Hvel et l. procedure ws 103.6% of the totl triglycerides originlly present in plsm, this ment tht 101% of the totl plsm cholesterol ws recovered by the rpid flottion procedure. As these determintions re within the 5.1% experimentl error (Methods section), the present results indicte tht, like cholesterol, the triglycerides were quntittively recovered from plsm by both flottion procedures. As with cholesterol, the triglyceride contents of frctions generted by the two flottion procedures were similr but not identicl. Both flottion procedures yielded similr mounts of triglycerides in the VLDL/IDL frction, s well s in the LDL frction (Tble 3). With the vribility described in Tble 3, only triglyceride difference of 8% (VLDL/IDL) nd 11% (LDL) would hve been detected with power of In contrst, the HDL frction isolted by the rpid flottion procedure hd significntly more triglyceride (9%) thn the HDL frction in the Hvel et l. procedure. The extr triglyceride represented only 1.0% (8.9% 10.8) of the totl triglyceride present in whole plsm. In conclusion, both the totl recovery nd distribution of triglyceride between the lipoprotein frctions were similr for both flottion procedures. The two flottion methods lso generted lipoprotein frctions with mtching triglyceride to cholesterol molr rtios (Tble 4). Closely resembling tht found in whole plsm, the rtio of summed recoveries confirmed tht neither flottion procedure produced differentil losses of the mjor neutrl lipids. Perhps more importntly, both flottion procedures generted HDL frctions with the sme molr rtio of triglycerides to cholesterol. Thus, the HDL isolted by the rpid flottion procedure TABLE 2. Lipoprotein recoveries s ssessed by GC MS quntittion of cholesterol TABLE 3. Lipoprotein recoveries s ssessed by enzymtic ssy of triglycerides Lipoprotein Frction Hvel et l. Flottion Procedure Rpid Flottion Procedure b Lipoprotein Frction Hvel et l. Flottion Procedure Rpid Flottion Procedure b VLDL/IDL % % LDL % % HDL % % c Summed cholesterol recovery from ll frctions % % Cholesterol contents were directly ssyed without concentrting or deslting the isolted frctions. Dt represent men percent SD (n 6) of the totl plsm cholesterol concentrtion (4.17 mm 0.96). b Dt represent men percent SEM (n 6) of cholesterol vlues in the corresponding Hvel et l. frction. c This vlue ws different (P 0.014) from tht found for the HDL cholesterol isolted by the Hvel et l. procedure (1). VLDL/IDL LDL HDL c Summed triglycerides recovered from ll frctions Triglyceride contents were directly ssyed without concentrting or deslting the isolted frctions. Dt represent men percent SD (n 6) of plsm triglyceride concentrtions (0.89 mm 0.52). b Dt represent men percent SEM (n 6) of triglyceride vlues in corresponding Hvel et l. frction. c This vlue ws different (P ) from tht found for HDL isolted by the Hvel et l. procedure. Tong, Knpp, nd VnRollins Isoltion of lipoproteins by sequentil flottions t 4 C 1701

7 TABLE 4. Triglyceride to cholesterol molr rtios in frctions isolted using Hvel et l. (1) nd rpid flottion procedures Lipoprotein Frction Hvel et l. Flottion Procedure did not hve selective increse in cholesterol reltive to triglycerides. DISCUSSION Rpid Flottion Procedure b VLDL/IDL LDL HDL (Summed triglycerides) (Summed cholesterol) Whole plsm Dt represent the men SD (n 6) of the triglyceride to cholesterol molr rtios. b Dt represent the men percent SEM. (n 6) of vlues in the corresponding Hvel et l. frction. In vivo oxidtions of the phospholipid, cholesterol, nd polipoprotein constituents of lipoproteins my ply n importnt role in the pthogenesis of therosclerosis (16, 19, 33, 34). To study lipoprotein oxidtions in vitro, the Hvel et l. (1) flottion procedure is the preprtive method most commonly used, nd requires severl dys for completion (Fig. 1). During this period, the polipoproteins my chnge or trnsfer mong different clsses of lipoproteins, lipoproteins my be degrded by enzymes or microorgnisms, nd lipid utooxidtion my occur (34). Recently, more rpid methods for prepring plsm lipoprotein frctions hve been proposed tht use tbletop ultrcentrifuges with higher g force cpcities (7 10). These brief reports described the detils of conditions to chieve fster seprtions; however, dt demonstrting the completeness nd reproducibility of the plsm lipoprotein seprtions were not presented. Moreover, most of the studies with tbletop ultrcentrifuges were crried out t room temperture, which my llow significnt utooxidtion to occur; free rdicls re redily formed from unsturted ftty cids t room temperture s well s t physiologicl temperture (35, 36). Recently, Cthcrt et l. (9) reported tht VLDL could be isolted in 2.5 h t 4 C using tbletop centrifuge nd rotor. However, they lso concluded tht the isoltion of VLDL under these conditions ws not very reproducible, with n RSD of 50% nd 34.2% for 0.38 nd 0.76 mm cholesterol, respectively. If one considers the temperture effects, the k fctors in the Hvel et l. (1) (k 90 ) nd Cthcrt et l. (9) (k 14) procedures, plus the time required to isolte VLDL in the Hvel procedure (20 h), it cn be clculted from k 1 /t 1 k 2 /t 2 tht t lest 4.3 h re required to isolte VLDL with the centrifuge nd rotor used by Cthcrt et l. In greement with these clcultions, we were unble to isolte VLDL t 4 C in less thn 4.0 h. To ensure reproducible isoltion of VLDL/IDL, we elected to routinely use 4-hr centrifugtions. In the present study, three lipoprotein frctions, VLDL/ IDL, LDL nd HDL, were isolted t 4 C in 4, 5, nd 9 h, respectively. The summed recovery of cholesterol nd triglycerides by this rpid flottion procedure ws essentilly identicl to tht determined with the Hvel et l. (1) method, nd ws very close to tht of whole plsm. In erly studies with the rpid flottion procedure, two fctors were found to be criticl for minimizing smple losses nd obtining quntittive recoveries. First, continued shrpness of the Teflon-covered blde in the tube slicer ws importnt becuse leks round the blde frequently developed within 10 to 15 cuts. Second, the inner surfce of the cut tubes nd the surfce of the blde hd to be thoroughly wshed fter ech trnsection. Close ttention to these two detils ws criticl in chieving quntittive recoveries. The distribution of lipoproteins, polipoproteins, protein, cholesterol, triglycerides, nd proteins mong rpidly floted lipoproteins ws similr to tht of the Hvel et l. method. Qulittively, the sme lipoproteins nd polipoproteins were present in comprble frctions subjected to electrophoresis; thus, the distribution of lipoproteins between the vrious frctions did not undergo mjor ltertions due to the use of very high centrifugl forces. However, 9 12% more triglycerides nd cholesterol were present in the rpidly floted HDL thn in the Hvel et l. floted HDL. It is possible tht this increse resulted from redistribution of smll mount of LDL to the HDL frction, becuse both frctions possessed similr triglyceride to cholesterol molr rtio (Tble 4). Moreover, Leonhrdt, Pietzsch, nd Nitzsche (37) noted tht use of n incresed centrifugl force decresed the recovery of LDL cholesterol by 9%. Although the mount of protein in comprble frctions ws similr for the two flottion procedures (Tble 1), the LDL frction contined five times more cholesterol per mg of protein thn did the HDL frction (Tbles 1, 2). Therefore, smll mount of LDL cholesterol nd triglyceride my hve been redistributed to the HDL frction, nd been ccompnied by n mount of polipoprotein too smll to be quntified (Tble 1) or visulized (polipoprotein A-I in Fig. 2). Alterntively, the incresed cholesterol nd triglyceride levels found my hve reflected n improved recovery of HDL in the rpid flottion procedure. During the isoltion of lipoprotein using the Hvel et l. flottion procedure, 2.8% of the totl cholesterol ws discrded from the LDL nd HDL infrntnts; in contrst, only 0.4% of the totl cholesterol ws discrded in the rpid flottions. Thus, 2.4% more cholesterol ws vilble to be isolted s HDL by the rpid flottion method. This difference ccounts for over two-thirds (8.4% 2.4/28.6) of the 12% increse in rpidly floted HDL (Tble 2). Thus, lthough both possibilities my hve contributed to the observed increse in HDL cholesterol nd triglycerides with the rpid flottion procedure, most of the increse ws probbly due to n improved recovery of HDL. Perhps more importntly, 9 12% increse in HDL lipids reflects less thn 1 4% of the cholesterol nd triglyceride levels in whole plsm Journl of Lipid Reserch Volume 39, 1998

8 TABLE 5. Comprison of cholesterol nd triglyceride concentrtions in lipoproteins isolted by flottion procedures Ref. VLDL/IDL LDL HDL 1.2 (g/ml) Summed Recovery Cholesterol Rpid flottion, n 6 plsms Hvel et l. flottion, n 20 ser Brousseu et l. flottion, n 10 ser 7 VLDL IDL Krpe et l. flottion, n 24 plsms Triglycerides Rpid flottion, n 6 plsms Brousseu et l. flottion, n 10 ser 7 VLDL IDL Krpe et l. flottion, n 24 plsms Dt represent men mm SD. Density g/ml. Concerning ccurcy, the rpid flottion procedure produced lipoprotein frctions with cholesterol concentrtions comprble to vlues published in the Hvel et l. study, s well s to those in more recent flottion procedures (Tble 5). The summed 97% totl recovery fell within the % rnge observed for these three studies. Moreover, the 59% LDL nd 31% HDL vlues were lmost identicl to the Hvel et l. vlues, nd within the 49 70% nd % rnge observed for helthy French nd Swedish volunteers (7, 38). Similr to our findings with the Hvel et l. procedure, Brousseu et l. (7) lso reported tht 4.4% of the totl cholesterol ws present in frction with density greter thn 1.2 g/ml. The 10% cholesterol in rpidly floted VLDL/IDL ws close to the corresponding Hvel et l. 13% vlue, nd between the % rnge reported in the two other studies. Thus, bsed on cholesterol determintions, the rpid flottion procedure produced resonble estimtes of lipoprotein concentrtions. The rpid flottion procedure lso generted lipoprotein frctions with triglyceride concentrtions comprble to recent published vlues (Tble 5; triglyceride concentrtions were not determined in the originl Hvel et l. study). A summed triglyceride recovery of 100% by the rpid flottion procedure ws within the % rnge observed for the two recent studies. Moreover, the 9.0% HDL nd 76% VLDL/IDL were comprble to the % nd 64% vlues observed for smples from helthy French nd Swedish volunteers (7, 38). However, the 15% LDL isolted by the rpid flottion procedure ws brely more thn hlf the 23 26% observed for Europen nd Swedish volunteers (7, 38). Thus, with the possible exception of the LDL frction, the rpid flottion procedure yielded lipoprotein frctions with resonble triglyceride distributions. In conclusion, the lipoprotein profiles derived by rpid flottions were very similr qulittively nd quntittively to those generted by the Hvel et l. procedure (1). Thus, by use of simple benchtop centrifuge, lipoproteins from up to 10.0 ml of plsm (or ten 1.0-ml plsm smples) could be isolted in 1 dy for chemicl chrcteriztion nd biologicl testing. Becuse lipoprotein utooxidtion nd redistributions during processing were not ssessed in the present study, future studies re needed to determine the extent to which rpid flottions t low tempertures reduce utooxidtion, nd whether specific lipid clsses, such s phospholipids with high levels of polyunsturted ftty cids, re protected. This work ws prtilly supported by n Americn Hert Assocition grnt, GIA , nd by NIH grnts HL-48877, HL , nd DK The uthors lso wish to cknowledge the helpful dvice of Dvid Chppel (University of Iow) regrding the qulittive nlyses. We would further like to cknowledge the College of Medicine Gs Chromtogrphy Mss Spectrometry Fcility for their grcious help. Mnuscript received 24 December 1997 nd in revised form 23 Mrch REFERENCES 1. Hvel, R. J., H. A. Eder, nd J. H. Brgdon The distribution nd chemicl composition of ultrcentrifuglly seprted lipoproteins in humn serum. J. Clin. Invest. 34: Schefer, E. J., S. Eisenberg, nd R. I. Levy Lipoprotein poprotein metbolism. J. Lipid Res. 19: Kunitke, S. T., nd J. P. Kne Fctors ffecting the integrity of high density lipoprotein in the ultrcentrifuge. J. Lipid Res. 23: Cstro, G. R., nd C. J. Fielding Evidence for the distribution of polipoprotein E between lipoprotein clsses in humn normocholesterolemic plsm nd for the origin of unssocited polipoprotein E (LpE). J. Lipid Res. 25: Brooks, C. J. W., R. M. Mckenn, W. J. Cole, J. McLchln, nd T. D. V. Lwrie Profile nlysis of oxygented sterols in plsm nd serum. Biochem. Soc. Trns. 11: Ry, B. R., E. O. Dvidson, nd H. L. Crespi Experiment on the degrdtion of lipoproteins from serum. J. Phys. Chem. 58: Brousseu, T., V. Clvey, J-M. Brd, nd J-C. Fruchrt Sequentil ultrcentrifugtion micromethod for seprtion of serum lipoproteins nd ssy of lipids, polipoproteins, nd lipoprotein prticles. Clin. Chem. 39: Leonhrdt, W., J. Pietzsch, U. Julius, nd M. Hnefield Recovery of cholesterol nd tricylglycerol in very-fst ultrcentrifugtion of humn lipoproteins in lrge rnge of concentrtions. Eur. J. Clin. Chem. Clin. Biochem. 32: Cthcrt, S., nd M. H. Dominiczk The mesurement of Tong, Knpp, nd VnRollins Isoltion of lipoproteins by sequentil flottions t 4 C 1703

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