Radioimmunoassay of Human Plasma Retinol-Binding Protein

Transcription

1 Rdioimmunossy of Humn Plsm Retinol-Binding Protein FRNK REES SMITH, MIRM RZ, nd DEWITT S. GOODMN From the Deprtment of Medicine, Columbi University College of Physicins nd Surgeons, New York 132 B S T R C T rdioimmunossy for humn plsm retinol-binding protein (RBP) hs been developed utilizing double ntibody precipittion technique. RBP ws purified 15- to 2-fold by procedures described previously. specific nti-humn RBP ntiserum ws prepred in rbbits by three once-weekly injections of purified RBP emulsified with Freund's djuvnt. RBP ws iodinted with 'I nd the RBP-"'I ws purified by gel filtrtion on Sephdex G-1 fter complex formtion with humn plsm prelbumin. The RBP-"'I ws completely (> 95%) immunoprecipitble in the presence of n excess of specific ntiserum, it ws not (< 5%) immunoprecipitble in the bsence of specific ntiserum, nd it could be completely displced from ntibody by excess unlbeled RBP. The stndrd curve obtined in the immunossy with norml plsm ws identicl to tht with pure RBP. Duplicte smples differed from their men by 5 ±+-5% (+SD). There ws quntittive recovery of pure RBP dded in vrying mounts to norml plsm. The immunossy ccurtely mesured RBP in mounts of 1-1 ng per ssy tube. There ws no significnt difference in the immunorectivity of po-rbp s compred to holo-rbp. The men plsm vlues (±SEM) for group of 76 norml subjects were 47.2 ±1.6 g/ml for mles nd 41.6 ±1.6 /sg/ml for femles. Plsm RBP levels were mrkedly depressed (15 ±2.3 gg/ml) in 14 ptients with cute virl heptitis. There ws highly significnt correltion between the plsm levels of RBP nd of vitmin in both norml subjects nd ptients with heptitis. In ll subjects plsm RBP ws generlly sturted wish retinol. The dt suggest tht under norml circumstnces RBP circultes lmost exclusively s the holoprotein. Doctors Smith nd Rz were Trinees under Grnts T1-M-5397 nd T1-M-5234 during this work. Dr. Goodmn is Creer Scientist of the Helth Reserch Council of the City of New York under Contrct I-399. Received for publiction 13 My 197. INTRODUCTION Vitmin circultes in humn plsm s retinol bound to specific protein, retinol-binding protein (RBP), which hs been isolted nd chrcterized recently in this lbortory (1). This protein hs l-mobility on electrophoresis, moleculr weight of pproximtely 21,, nd single binding site for one molecule of retinol. In plsm, RBP circultes together with plsm prelbumin (P), s protein-protein complex. Previously, on the bsis of the content nd recovery of protein-bound retinol during RBP purifiction, it ws suggested tht the usul concentrtion of RBP in plsm is of the order of 3-4,g/ml of plsm (1). We report the development of rdioimmunossy for RBP in humn plsm. With this imunossy, the level of RBP in series of norml subjects nd of ptients with heptitis hs been determined. The correltion between plsm RBP nd vitmin levels hs lso been exmined. METHODS Purifiction of RBP. RBP nd P were isolted from whole humn plsm (usully obtined s outdted plsm from the blood bnk) by the following sequence of procedures: chromtogrphy on DEE-Sephdex, gel filtrtion on Sephdex G-2, repet DEE-Sephdex chromtogrphy, preprtive polycrylmide gel electrophoresis, nd gel filtrtion on Sephdex G-1. These procedures hve been described in detil in our previous publictions (1-3). Preprtion of ntiser. solution of RBP, 6 mg/ml in.1 M N phosphte buffer ph 7.4, ws emulsified with n equl volume of complete Freund's djuvnt.' White rbbits weighing pproximtely 2 kg were injected three times, t weekly intervls, with.4 ml of the emulsified RBP (.1 ml per toe pd). 1 month fter the first injection the nimls were exsnguinted by crdic puncture; fter clotting, the blood smples were centrifuged t 2 rpm for 3 min t 4 C nd the ser were collected. The serum from the niml with the highest titer ginst RBP (precipitin line by immunodiffusion (4) ginst 1: 8 dilution of norml plsm) ws used in the immunossy. control ntiserum ws prepred by injecting rbbits in similr fshion with humn 1-cid glycoprotein puri- 1 Difco Lbortories, Detroit, Mich The Journl of Clinicl Investigtion Volume

2 4 ±- 2 E.I co 1 I- N 4 x -J Ix n 3 41 I-. RBP-'31 + P. CL 1 o FRCTION NUMBER FIGURE 1 Purifiction of RBP-1"I by gel filtrtion on columns of Sephdex G-1, first together with humn serum lbumin (HS, top pnel), nd then together with prelbumin (bottom pnel). See text for detils. fied in our lbortory (2). This ntiserum showed precipitin lines on immunodiffusion when tested ginst humn 1-cid glycoprotein or humn serum lbumin. The ntiserum showed no precipitin lines when tested ginst purified RBP. Iodintion; purifiction of RBP-13'I. RBP ws iodinted with 'I using the ICi method of McFrlne (5) s modified by Wldmnn.2 Lyophilized RBP (1 mg) ws dissolved in 1 ml of.4 M N borte buffer ph 8. nd dded to vil contining high specific ctivity (35-5 mci//g) crrier-free N-'31I 3 previously mixed with n equl volume of.1 N HCl..33 M IC1 ws diluted 1:1 with 2 M NCl. n mount of the dilute ICI solution (usully 6 jul) ws then dded to the vil so s to pproximte n equimolr rtio between l'i nd RBP ssuming 2% efficiency of iodintion. The observed efficiency of iodintion s clculted from recovery of 'I s lbeled RBP fter purifiction ws usully close to 35%. Immeditely fter dding the IC1, the solution ws mixed nd pplied to Sephdex G-5 column (16 X.8 cm) previously wshed with 2 ml of humn serum lbumin' (1.2 mg/ml) in.4 M N borte buffer ph 8.. The proteins were eluted with borte buffer t room temperture; frctions of.5 ml were collected in vils contining 4 mg of humn serum lbumin ech nd 2Wldmnn, T.. Personl communiction. 'Iso/Serve, Cmbridge Nucler Corp., Cmbridge, Mss. 'Proserum, Dow Chemicl Co., Midlnd, Mich. then ssyed for '"I. The mjor portion of the rdioctivity ws eluted with RBP immeditely fter the void volume. RBP-'I ws purified by gel filtrtion on Sephdex G-1 before nd fter complex formtion with P. The frctions from the Sephdex G-5 column contining the first hlf of the RBP-31I pek (pproximtely.5 mg of RBP in 2 ml) were pooled nd were then chromtogrphed on Sephdex G-1 column (8 X 1.4 cm) t 4VC, using.2 M K phosphte ph 7.4 contining.2 M NCl s eluting buffer, with flow rte of 15 ml/hr. The effluent ws monitored continuously t 28 nm with n LKB' Uvicord II bsorptiometer; frctions of 6 ml were collected nd ssyed for 'I. Rdioctivity ws recovered in n effluent volume chrcteristic of RBP (moleculr weight pproximtely 21,) (Fig. 1, top). The frctions contining RBP-wI (frctions of Fig. 1, top) were pooled nd concentrted to 2 ml by ultrfiltrtion using n micon' UM-1 filter. Lyophilized humn prelbumin, 4 mg, ws dissolved in the RBP-'I solution; fter 15 min t room temperture, the solution ws chromtogrphed on column of Sephdex G-1 of similr size nd under identicl conditions % of the rdioctivity ws now eluted in volume chrcteristic of the RBP-P complex (Fig. 1, bottom, frctions 1-14). The solution of purified RBP-'I (in the form of complex with P) ws stored in the drk t - 2'C until used. 'LKB Instruments, Inc., Rockville, Md. 'micon Corp., Lexington, Mss. Rdioimmunossy of Humn Plsm Retinol-Binding Protein 1755

3 16 w,_ 12 wz-. Z 48 w V L I 2 2 LBUMIN = P4 BUFFER = P4 BUFFER + SILICONE o = BRBITL * = BRBITL + SILICONE 2 3 (g/1ml) FIGURE 2 Effect of ssy conditions on the precipittion of RBP-'1I in the bsence of nti-humn RBP ntiserum..5 N brbitl buffer ph 8.5 in glss tubes, ;.5 brbitl ph 8.5 in siliconized tubes,*;.5 K phosphte buffer ph 7.7 in glss tubes, ;.5 phosphte ph 7.7 in siliconized tubes,. 4 Studies crried out fter the development of the rdioimmunossy procedure demonstrted tht n equivlent purifiction of the RBP-312I could be chieved by omission of the first Sephdex G-1 gel filtrtion (of RBP-'I plus serum lbumin). In most of the studies reported here the usul procedure ws to crry out single Sephdex G-1 chromtogrphy fter complex formtion between the lbeled RBP nd P. Immunossy procedure. rdioimmunossy ws developed using double ntibody precipittion technique, generlly similr to the method of Morgn nd Lzrow for the immunossy of insulin (6). Incubtion conditions were selected to reduce the nonspecific precipittion of RBP-I I observed in the bsence of the specific ntiserum ginst RBP. With.5 m brbitl buffer ph 8.5 contining 1% humn or bovine serum lbumin in untreted glss test tubes, the nonspecific RBP-I I precipittion ws 1% (Fig. 2) nd this ws chosen s the buffer system (designted brbitllbumin buffer). In ech ssy,.1 ml of diluted rbbit ntiserum (including both the specific nti-rbp nd the control ntiser) tion tmier tubes8 nd the plsm ws seprted by centrifug- t ws dded to 75 X 1 mm disposble test tube, followed in order by stndrd REP or humn plsm diluted in brbitllbumin buffer, brbitl-lbumin buffer lone, nd finlly RBP-I I; the finl volume of the ssy mixture ws.5 ml. The sequence of dding regents ws lwys the sme. The mixture of nti-rbp ntiserum nd control ntiserum ws usully prepred so tht the finl dilution of the nti-rep ntiserum in the ssy tube ws 1: 5 to 1: 6, wheres the finl dilution of the control ntiserum ws 1: 4. This mount of control ntiserum ws dded in order to ensure uniform nd nerly complete recovery of rbbit gmm globulin in the finl precipitte. The totl mount of rbbit gmm globulin ws identicl in ll smples. t this dilution of the nti-rep ntiserum, 5-6% of the REP- 3I ws usully ntibody bound in the bsence of dded unlbeled REP. fter prepring the ssy mixtures, the tubes were put in the drk t 4C for overnight (18 hr) incubtion. In the morning.1 ml of nti-rbbit gmm globulin ntiserum, mde in gots,' ws dded to ech tube, nd 7Pentex Eiochemicl, Knkkee, Ill F. R. Smith,. Rz, nd DeW. S. Goodmn the tubes were mixed nd returned to the drk t 4C for 135 min; the second incubtion produced nerly quntittive precipittion of rbbit gmm globulin. fter the second incubtion, the precipittes were collected by centrifugtion t 4VC for 15 min t 17,3 g in Sorvll model RC-2B centrifuge. The superntnt solutions were removed, the precipittes wshed with.5 ml of brbitl-lbumin buffer, nd the wsh nd superntnt solutions combined. The smples were then ssyed for "'1I in Pckrd utogmm spectrometer. In the ssy, 'I found in the precipitte represented ntibody-bound RBP-'31I, wheres "2I in the superntnt-wsh solution represented free (i.e. not bound to ntibody) RBP. fter rdiossy, the bound/free rtio of RBP-'3I ws clculted for ech smple, nd the mount of RBP in the ssy tube ws then determined from the stndrd curve for tht experiment. Immunossy controls. In ech set of ssys, tubes were included in which () the specific nti-rbp ntiserum ws used t finl dilution of 1: 4 (without the ddition of control ntiserum) (b) the specific nti-rbp ntiserum ws omitted from the ssy mixture (control ntiserum lone t finl dilution of 1: 4), nd (c) lrge excess (1-5,ug) of unlbeled RBP ws dded to the usul ssy mixture. RBP stndrd. solution of purified RBP, 2-3 /Lg/ml in H2, ws kept frozen in the drk. Just before use the solution ws thwed; n mount contining,ug 1 RBP ws removed, mixed with of solution contining lug 4 of purified P, nd then djusted to 1. ml volume with brbitl-lbumin buffer. This solution of RBP (1 ttg/ml) ws diluted serilly with brbitl-lbumin buffer to concentrtions of,ug/ml 1 nd.1 tg/ml nd these solutions were used to determine the stndrd curve. The concentrtion of RBP in the stock stndrd solution ws checked ech time before mking the first dilution by determining the ultrviolet bsorption spectrum of the solution. stndrd curve ws determined with ech set of ssys nd ws used to clculte the levels of RBP in tht prticulr set of smples. Lter experiments hve shown tht the ddition or omission of purified P to the purified REP does not ffect the vlues obtined in the stndrd curve. Plsm smples. Smples were obtined from men nd women with norml medicl histories nd physicl exmintions. Femle subjects were not tking contrceptive mediction ml of blood ws collected in heprinized vcu- 4 C, frozen, nd stored in the drk. Before use the smples were thwed nd diluted 1:1 to 1: 2'with brbitl-lbumin buffer. Plsm dilutions were selected so tht the bound/free rtios for RBP-12I1 fell on the most sensitive portion of the stndrd curve (see below). Smples of plsm were obtined lso from 14 ptients with cute virl heptitis during the erly phse of their illness (men plsm bilirubin, 11.9 mg/1 ml; serum glutmic oxlocetic trnsminse [SGOT], 1613 mu/ml; nd lkline phosphtse, 162 mu/ml).. The smples were prepred nd ssyed in the sme mnner s those from norml subjects. Vitmin determintions. Plsm vitmin levels were mesured by the trifluorocetic cid method of Dugn, Frigerio, nd Siebert (7) s modified by Roels nd Mhdevn (8). Sttistics. Correltion coefficients were clculted on n Olivetti Progrmm 11 1 using progrm prepred for this 8 Becton-Dickinson Co., Rutherford, N. J. 'Norm SGOT 1-5 mu/ml nd lkline phosphtse 3-85 mu/ml. 1 Olivetti Underwood Corp., New York.

4 instrument (9). Significnce ws ssessed s outlined by Snedecor nd Cochrn (1). RESULTS nti-humn RBP ntiserum. The ntiserum obtined from rbbits which hd been injected with purified RBP gve single precipitin line when tested ginst purified RBP by either immunodiffusion (4) or immunoelectrophoresis (11). When the ntiserum ws tested ginst norml humn whole plsm, however, three precipitin lines were obtined. The mjor line showed rectionof-identity with the single line obtined with purified RBP; one of the two minor lines ws identified s immunoprecipitted humn serum lbumin. The ntiserum thus contined ntibodies ginst two humn plsm proteins in ddition to those ginst RBP. The presence of these dditionl ntibodies in the ntiserum in no wy interfered with the precision nd specificity of the RBP rdioimmunossy since the ntiserum showed only single line ginst purified RBP nd since the 'I lbel ws ttched only to purified RBP. This prticulr ntiserum ws used in the studies reported here. More recently, pure nti-humn RBP ntiserum ws prepred by rechromtogrphy of purified RBP on column of Sephdex G-1, before the preprtion of the RBP-Freund's djuvnt emulsion nd immuniztion of rbbits s described bove. single precipitin line ws obtined by testing this ntiserum ginst whole plsm, nd this line showed rection-of-identity with the line obtined by recting this ntiserum ginst purified RBP (Fig. 3). Stndrd curve. Fig. 4 shows the curve describing the displcement of RBP-'I from ntibody by incresing mounts of unlbeled pure RBP. The curve ws sigmoid in ppernce on semilogrithmic plot, with the most sensitive portion of the curve corresponding to the ddition of 1-5 ng of RBP per ssy tube. ssy of humn plsm. Plsm from single subject ws diluted 1: 1 nd 1:1 with brbitl-lbumin buffer nd ssyed t vrious dilutions simultneously with stndrd RBP. s shown in Fig. 4, the immunossy curve obtined with whole plsm ws identicl in shpe with the curve obtined with pure RBP. This demonstrted tht RBP in whole plsm displced RBP-'I from ntibody in mnner which ws quntittively identicl to the displcement obtined with purified RBP. Recovery of RBP. Norml plsm ws diluted with brbitl-lbumin buffer to concentrtions of pproximtely 9-15 ng of RBP per.1 ml. Pure RBP ws diluted to concentrtions of.1 nd 1. /g/ml, nd portions of these solutions were dded to the plsm smples so s to enrich the RBP content by eight mounts rnging from increments of 1 to 25%. Ech smple ws then ssyed in triplicte. The men recovery +SEM of FIGURE 3 Immunodiffusion study of RBP ntiserum ginst purified RBP plsm (WP). rbbit nti-humn nd humn whole the pure RBP dded to the plsm smples ws %. The recovery of pure RBP in the ssy ws determined by subtrcting the RBP vlues obtined with plsm lone from the vlues obtined with the smples enriched with known mounts of pure RBP. Immunossy controls. When reltively lrge mount (dilution 1: 4) of specific nti-rbp ntiserum ws dded to the ssy tube, 95-1% of the dded 'I ws recovered in the immunoprecipitte. This demonstrted tht the 'I lbel ws virtully completely ttched to immunorective RBP nd tht no "dmge" to the lbeled ntigen hd occurred during the first incubtion. In contrst, when the specific nti-rbp ntiserum ws omitted from the ssy, less thn 5% of the 'I ws found in the precipitte. Finlly, more thn 95% of the RBP-'I could be displced from the specific ntibodies by the ddition of 2 iyg of unlbeled pure RBP. These results estblished the specificity of the rdioimmunossy for RBP, nd such controls were run with ech set of ssys. If the control vlues differed from those just described, the ssy results obtined in tht experiment were discrded. Intr-ssy (within ssy) greement. Plsm smples were routinely ssyed in duplicte, nd the men of the pir of vlues tken s the finl vlue. During n 8 month period the vlues for duplicte ssys on 73 smples from norml subjects differed from the men vlue for ech duplicte pir by n verge of 5.4 ±5.% (+SD). On the bsis of these dt, the results for smple were discrded when duplicte ssys differed Rdioimmunossy of Humn Plsm Retinol-Binding Protein 1757

5 & s s 1.2 w w Or \ 4 %,. & ~~~l_. 5 o 2 5 l 2 5icooo 5 RBP (ng) FIGURE 4 The RBP rdioimmunossy stndrd curve. The figure demonstrtes the displcement of RBP-.4I from ntibody by purified RBP nd by dilutions of norml plsm. The points for norml plsm were determined by first clculting the men vlue for the level of RBP in the smple of plsm from the severl ssys crried out on tht smple. The expected mount of RBP dded per ssy tube ws then clculted from the mount of diluted plsm dded. In the figure these vlues for RBP re plotted ginst the observed bound/free rtios for the corresponding smples o s PURE RBP E X NORML PLSM. O.6 W o 6\o \6 z m sists of three bnds, two fluorescent nd one nonfluorescent, ll with,-mobility (3). Evidence hs been presented (3) which suggests tht during purifiction the nturlly-occurring form of RBP ("H2" form) is grdu 'IO from ech other by more thn 2%, nd the ssy of tht smple ws repeted. Interssy (between ssy) greement. smple of plsm from single subject ws divided into 2-ml portions nd stored frozen nd in the drk. During 6 month period, portions were ssyed in duplicte in 14 different sets of ssys. The men vlue +SD for the smple ws 47.1 ±3.8 ig of RBP per ml. ssy of lipoproteins nd of proteins with density (D) > Since retinol is lipid-soluble vitmin, the question rose s to whether there might be ny immunologic cross-rectivity between RBP nd the lipoproteins responsible for the trnsport in plsm of most of the other plsm lipids. Plsm smples from three subjects were djusted to hydrted density of 1.21 by the ddition of solid KBr, nd the lipoproteins were seprted en bloc by ultrcentrifugtion for 4 hr t 114, g (12). The lipoprotein (D < 1.21) nd the D > 1.21 protein frctions were dilyzed overnight ginst isotonic NCl. Immunossy for RBP of the lipoprotein smples, t concentrtion 1.5 times tht found in whole plsm, reveled no immunorectivity. The D > 1.21 protein frctions, in contrst, were fully immunorective, with complete recovery of the RBP immunorectivity found in the unfrctionted whole plsm. These ltter results were expected since RBP hs hydrted density greter thn 1.21 (1). Comprison of holo-with po-rbp nd of RBP subspecies. Purified RBP s isolted in our lbortory is microheterogeneous on disc-gel electrophoresis nd con F. R. Smith,. Rz, nd DeW. S. Goodmn

6 lly converted to more rpidly migrting form of the holoprotein ("HI") nd, in turn, to the retinol-free poprotein ("" form). Two experiments were crried out in order to compre the immunorectivity of holo-rbp with tht of po- RBP nd of the different RBP subspecies with ech other. In the first experiment smple of purified RBP ws lrgely converted from holo-rbp to po-rbp by extrction of the protein-bound retinol into heptne (13). The bsorbnce rtio (33 nm/28 nm) of the solution of RBP ws.79 before extrction nd.16 fter extrction, indicting tht 8% of the protein-bound retinol hd been extrcted into heptne. Portions of the unextrcted (minly holo) nd of the extrcted (minly po) RBP smples were ssyed simultneously, nd the results re shown in Fig. 5. There ws no significnt difference in the immunorectivity of the two preprtions. In the second experiment, the immunorectivity of smple of RBP consisting of mixture of ll three bnds (minly "H2" + "HI") ws compred with the immunorectivity of smples of the lmost pure "" nd "HI" forms, respectively (see disc-gel ptterns Nos. 1, 2, nd 3 in Fig. 2 of reference 3 for the ppernce on electrophoresis of the three smples). The "" nd "Hi" smples were prepred by repet preprtive.4 _ C.4 - m R BP (ng) FIGuim 5 Displcement of RBP-I from ntibody by purified (minly holo) RBP nd by extrcted (po) RBP. ) IL F.6 F O "--O R8P BNDS - H2 +HI (+) O- HI OO y I s RBP (ng) \,& ~~~ 56 FIGURE 6 Displcement of RBP-II from ntibody by different RBP subspecies. polycrylmide-gel electrophoresis of smple of purified RBP (3). It should be noted tht we hve previously shown tht the "" nd "Hi" forms (previously designted RBP. nd RBPh [1]) hve identicl mino cid compositions. The results shown in Fig. 6 indicte tht the different RBP subspecies hve similr immunorectivities in the immunossy. Plsm RBP levels. The men plsm RBP level (±-SD) for 38 norml mle subjects ws 47.2 ±9.6 jg/ ml nd for 38 norml femles ws 41.6 ±1. /g/ml. There ws no correltion between plsm RBP concentrtion nd ge in either group. The plsm RBP level ws mrkedly depressed in the 14 smples from ptients with cute virl heptitis ( isg/ml, men ±SD). Fig. 7 shows the plsm levels of RBP nd of vitmin for the norml subjects nd the ptients with heptitis. Correltion of RBP nd vitmin. The level of plsm RBP ws significntly correlted with the level of plsm vitmin in both the group of 76 norml subjects (r =.382, P <.1) nd the group of 14 ptients with heptitis (r =.745, P <.3). Fig. 7 shows the regression line for the combined vlues from both the norml nd the heptitic subjects (r =.692, P <.1). Rdioimmunossy of Humn Plsm Retinol-Binding Protein 1759

7 z - 8 qm.:k -J. sof 4 F 6-2- o m O I * e k "'-I NORML MLES - NORML FEMLES *E CUTE HEPTITIS PLSM RBP (Mug/mi) FIGURE 7 Correltion between plsm RBP nd plsm vitmin concentrtions in norml mles, norml femles, nd subjects with cute heptitis. The reltionship between plsm RBP nd vitmin levels cn lso be exmined by estimting the extent to which RBP in plsm is sturted with retinol. Since ech RBP molecule cn bind nd trnsport single molecule of retinol, it ws possible to clculte redily the level of plsm retinol which would be expected for complete sturtion of the protein t ny given level of RBP. The expected retinol level for complete sturtion ws then compred with the observed vitmin level for ech smple of plsm. In the norml mle subjects the men sturtion of RBP with retinol +SD ws 88 ±2% nd in the femle subjects, 82 ±26%. The extent of RBP sturtion could not be estimted with comprble relibility in the subjects with heptitis becuse of the lrge reltive error inherent in the mesurement of very low levels of vitmin in plsm. The dt (see Fig. 7) suggest, however, tht RBP ws fully sturted with retinol in these ptients. DISCUSSION rdioimmunossy hs been developed to mesure ccurtely the level of RBP in humn plsm. The immunossy is specific for RBP nd hs firly high degree of quntittive precision (reproducibility) nd relibility. The specificity of the rdioimmunossy ws studied by demonstrting tht the RBP-'I ws completely (> 95%) immunoprecipitble in the presence of n excess of specific ntiserum, tht it ws not (< 5%) immunoprecipitble in the bsence of specific ntibody, nd tht the RBP-1I could be lmost completely displced from ntibody by excess unlbeled purified RBP. 176 F. R. Smith,. Rz, nd DeW. S. Goodmn Control tubes exmining ech of these chrcteristics were included with ech set of ssys. Smples were routinely ssyed in duplicte, with individul vlues differing by 5 ±5% (men ±SD) from the men vlue for ech duplicte pir. The repeted nlysis of single smple of plsm during 6 month period gve vlues vrying from their men by ±8% (SD). The results pper to be relible since there ws quntittive recovery of purified RBP dded in vrying mounts to norml plsm. The immunossy ccurtely mesures RBP in the mounts of 1-1 ng per ssy tube. The sensitivity of the ssy is such tht norml plsm must be extensively diluted before ssy with the ddition of the equivlent of pproximtely 1 4 of plsm per ssy tube. It ws reported previously tht purified RBP did not rect with commercil ntiser ginst whole humn serum or ginst vriety of individul humn serum proteins including prelbumin (1). The finding tht RBP filed to rect with nti-humn whole serum ntiserum suggested tht RBP might be reltively poor ntigen compred to most other serum proteins. The results presented here demonstrte, however, tht purified RBP is n effective ntigen nd tht n ntiserum specific for RBP cn be prepred in rbbits by conventionl procedures. RBP ppers to be immunologiclly distinct from other known serum proteins. s discussed bove, purified RBP is microheterogeneous on disc-gel electrophoresis nd consists of three bnds, two fluorescent nd one nonfluorescent (3). Since the purified RBP used for immunizing rbbits, for iodintion, nd for stndrd solutions in the immunossy ws mixture of these three components, it ws importnt to determine whether the subspecies differed in their immunorectivity in the ssy. If there were significnt vrition, for exmple, the ssy might not mesure ccurtely the totl level of RBP in subjects with vrying proportions of holo- nd poprotein circulting in their plsm. The immunorectivity of po- RBP, prepred by heptne extrction of retinol from RBP or isolted fter prolonged hndling nd purifiction, ws compred with tht of the stndrd RBP s well s with tht of the isolted holo-rbp subspecies. There were no significnt differences in the immunorectivity of the po- s compred to the holo-rbp or mong the different RBP subspecies. It ws previously suggested (3) tht during purifiction the nturlly occurring form of holo-rbp is converted to more rpidly migrting form of the holoprotein nd, in turn, to the poprotein. The results presented here indicte tht the totl mount of RBP in plsm would be determined ccurtely in the immunossy regrdless of the proportions of the different subspecies present t the time of ssy.

8 Using the rdioimmunossy, the men plsm RBP vlues (±SEM) for group of 76 norml subjects were 47.2 ±1.6 g/ml for mles nd 41.6 ±1.6 /g/ml for femles. The femles were not on contrceptive mediction, nd there ws no correltion between RBP levels nd ge in these groups. The men RBP level in this group of mles ws significntly higher thn in the femles. Ptients in the erly phse of cute virl heptitis showed mrked depression of their plsm RBP levels. It is likely tht this depression resulted from decresed rte of RBP synthesis nd secretion by the disesed liver. lthough direct evidence for the synthesis nd secretion of RBP in the liver is not vilble, indirect evidence for this conclusion includes the fct tht vitmin is minly stored in (nd cn be mobilized redily from) the liver (14), s well s these findings in ptients with heptitis. Studies re currently in progress to further define the effects of liver disese on the levels nd metbolism of RBP nd to exmine the pthophysiologicl consequences of such effects. highly significnt correltion ws found between the plsm levels of RBP nd of vitmin in both the helthy subjects nd the ptients with heptitis. The bsolute levels of RBP nd vitmin were such tht the two components were generlly present in nerly equimolr mounts with men molr rtio (vitmin /RBP) in the norml subjects of.85. The findings suggest tht smll mount of po-rbp my normlly circulte in plsm in some subjects, lthough the combined bsolute errors in the methods for ssying RBP nd vitmin mke this conclusion n uncertin one. The results indicte, however, tht in this group of subjects plsm RBP ws generlly sturted with retinol, nd the dt suggest tht under norml circumstnces RBP circultes lmost exclusively in the form of the holoprotein. CKNOWLEDGMENTS We thnk Mr. T. Shirtori nd Miss E. Miller for expert ssistnce. We re grteful to Doctors. G. Frntz, C. L. Christin, J. H. Morse, nd L. J. Kgn for dvice nd discussions. This work ws supported in prt by Grnt M-5968 from the Ntionl Institutes of Helth nd by Contrct No. DD 17-7-C-2 from the Deprtment of the rmy. REFERENCES 1. Kni, M.,. Rz, nd DeW. S. Goodmn Retinol-binding protein: the trnsport protein for vitmin in humn plsm. J. Clin. Invest. 47: Rz,., nd DeW. S. Goodmn The interction of thyroxine with humn plsm prelbumin nd with the prelbumin-retinol-binding protein complex. J. Biol. Chem. 244: Rz,., T. Shirtori, nd DeW. S. Goodmn Studies on the protein-protein nd protein-lignd interctions involved in retinol trnsport in plsm. J. Biol. Chem. 245: Ouchterlony, Diffusion-in-gel methods for immunologicl nlysis. In Progress in llergy. P. Kllos nd B. H. Wksmn, editors. S. Krger G, Bsel. 6: McFrlne,. S Metbolism of plsm proteins. In Mmmlin Protein Metbolism. H. N. Munro nd J. B. llison, editors. cdemic Press, Inc., New York. 1: 331. ppendix XIV. 6. Morgn, C. R., nd. Lzrow Immunossy of insulin: two ntibody system. Plsm insulin levels of norml, subdibetic nd dibetic rts. Dibetes. 12: Dugn, R. E., N.. Frigerio, nd J. M. Siebert Colorimetric determintion of vitmin nd its derivtives with trifluorocetic cid. nl. Chem. 36: Roels, O., nd S. Mhdevn Vitmin. In The Vitmins. P. Gyorgy nd W. N. Person, editors. cdemic Press, Inc., New York. 2nd edition. 6: Olivetti Underwood Progrmm 11, Sttisticl nlysis Mnul J. B. Willims, editor Snedecor, G. W., nd W. G. Cochrn Sttisticl Methods. Iow Stte University Press, mes. 6th edition Grber, P., nd C.. Willims, Jr Methode Immuno-Plectrophoretique D'nlyse de melnges de substnces ntigeniques. Biochim. Biophys. ct. i7: Hvel, R. J., H.. Eder, nd J. H. Brgdon The distribution nd chemicl composition of ultrcentrifuglly seprted lipoproteins in humn ser. J. Clin. Invest. 34: Goodmn, DeW. S Retinol trnsport in humn plsm. mer. J. Clin. Nutr. 22: Moore, T Vitmin. Elsevier Publishing Compny, New York. 28. Rdioimmunossy of Humn Plsm Retinol-Binding Protein 1761