Liquid-ChromatographicAssay for Free and Transthyretin-BoundRetinol-BindingProteinin Serum from Normal Humans

Transcription

1 CLIN. CHEM. 35/4, (1989) Liquid-ChromtogrphicAssy for Free nd Trnsthyretin-BoundRetinol-BindingProteinin erum from Norml Humns Betty Jne Burn nd Mrk & Kutnlnk We quntified vitmin A-trnsporting retinol-binding protein (RBP) in serum or plsm by size-exclusion high-performnce liquid chromtogrphy, using TK 2 column nd fluorescent detection of the bound retinol. erum or plsm smples filtered through.2-sm (pore size) Millex filter were pplied directly to the column. The ph 7. mobilephse contined sodium phosphte, disodium EDTA, nd mercptoethnol. Two peks with RBP immunologicl ctivity were eluted: the smller pek contining t lest 86% of the vitmin A, which ws identified s trnsthyretin-bound RBP; the lrger pek contining smll mount (<14%) of highly fluorescent vitmin A-contining protein, identified s free RBP. Both free nd trnsthyretin-bound RBP cn be quntified by this method. AddItIonl Keyphrees: retinoids rdil immunodiffusion fluorometty - vitmin A chromtogrphy, reversed-phse chromtogrphy, size-exclusion prelbumin rts Retinol-binding protein (RBP) is the mjor trnsport protein for retinol in serum (1). Usully, RBP circultes in blood in n equiinolr complex with trnsthyretin (prelbumm) (1). Totl RBP concentrtion in blood cn be used to detect retinol (vitmin A) deficiencies in nimls nd humns (2,3). However, totl RBP is lso influenced by liver disese nd protein-clorie mlnutrition (4, 5) nd is not highly correlted with body stores of vitmin A except in deficiency. Recently, Fex nd Felding (6) reported tht low vlues for free holo-rbp (RBP bound to retinol, but not to trnsthyretin) correlted well with bnorml drk dpttion (n indictor of vitmin A deficiency in lcoholics). However, they did not ctully mesure free RBP, but clculted it from the reltive mounts of RBP nd trnsthyretin in serum. The methods currently used for ssying RBP re: rdil immunodiffusion (RID) (7), rdioimmunos8y (8), lser immunonephelometry (9), enzymoimmunossy (1), turbidimetry (11), enzyme-linked immunosorbent ssy (12), electrophoresis with detection by fluorometric densitometry (13), nd electrophoresis followed by isoelectric focusing nd immunoblotting (14). All these methods hve limittions. RID, currently the most populr method becuse of its ese of use, nd ll other immunology-bsed techniques re species specific (1, 15), mking comprisons between niml nd humn dt difficult. Moreover, ll RBP is quntified, whether or not it ctully crries vitmin A, nd immunologicl techniques cnnot quntify both free nd trnsthyretin-bound RBP esily, becuse ntibodiesto RBP displce trnsthyretin from the complex (1, 16). Upon electrophoresis, RBP seprtes into microheterogeneous bnds, mking quntific- UDA/AR/PWA, Western Humn NutritionReserchCenter, Biochemistry Reserch Unit, P.O. Box 29997, Presidio of n Frncisco, CA Received December 1, 1988; ccepted Jnury 19, tion difficult. Also, the combined use of electrophoresis, isoelectric focusing, nd immunoblotting is too expensive nd time consuming for mss-popultion screening. Here we describe size-exclusion high-performnce liquid-chromtogrphic (HPLC) nlysis for free nd trnsthyretin-bound holo-rbp in serum nd plsm, with use of TK 2 column nd fluorometric detection. mple preprtion is miniml: we filter the serum through.2- pm Millex ifiter. Only vitmin A-contining RPB (hole- RBP) is mesured, nd rt nd humn RBP cn be quntifled under identicl conditions. Mterils nd Methods Apprtus. RBP ws ssyed with eries 4 liquid chromtogrph equipped with n LCI 1 computing integrtor nd n I 1 utosmpler (ll from Perkin-Elmer Corp., Mountin View, CA). Detection ws with Krtos F 98 fluorometer (ABI Anlyticl, Rmsey, NJ) t n excittion wvelength of 33 nm, with 418-nm emission filter. The column used ws 3 cm x 7.5 mm Altex pherogel TK 2 nlyticl column with 7.5 cm x 7.5 mm Altex pherogel TK precolumn (Beckmn Instruments, Inc., Fullerton, CA). The excittion nd emission spectr of the peks eluted from the HPLC column were determined with Perkin-Elmer L-3B fluorescence spectrometer. Fluorescent nd ultrviolet pek res were compred by ttching second LCI 1 computing integrtor nd Perkin-Elmer LC 9 UV-Vis spectrophotometric detector to the liquidchromtogrphy system. Totl plsm or serum retinols, nd retinol extrcted from TK HPLC peks, were quntified with Model 18 HPLC equipped with C18 reversed-phse column nd Model 14 diode rry UV-Vis spectrophotometer (both from Hewlett-Pckrd, unnyvle, CA). Chemicls nd regents. RBP stndrd for RID pltes, purified humn trnsthyretin, nd RID pltes for RBP nd trnsthyretin were from Behring Dignostics, n Diego, CA. Free RBP (purified from humn urine) ws from Clbiochem, L Joll, CA. Retinyl cette ws from igm Chemicl Co., t. Louis, MO. All other regents nd chemicls were HPLC or regent grde. HPW of retinol-binding protein. Humn plsm smples were obtined from helthynonsmoking dultvolunteers: 26 men nd 12 women, ges yers. Blood ws collected from ech subject on severl different dys of ech study.mples from 12 of the men were nticogulted with citrte, the others with heprin. The subjects were fed diets contining n verge of 5 int. units of vitmin A per dy. Blood ws lso obtined from the til veins of six helthy, chow-fed, dult, mle prgue-dwley rts nd the heprinized plsm ssyed. Plsm or serum ws diluted fourfold with isotonic sline (15 mmol/l NC1), ifitered through.2-pin (pore size) ifiter, nd 1 pl ws injected onto the TK column. The ph 7. mobile phse for isocrticelution of the smple consisted of,per liter, 15 mmol of sodium phosphte, CLINICAL CHEMITRY, Vol. 35, No. 4, 1989

2 mmol of disodium EDTA, nd 2 mmol of mercptoethnol. The flow rte ws.5 ml(min. We identified peks by compring their retention times with those of purified RBP, trnsthyretin, nd protein stndrds used for moleculr-mss clibrtion; by extrcting retinol from HPLC peks; nd by compring the results with RID plte results for RBP nd trnsthyretin in humn smples (Tble 1). HPLC of retinol in plsm. Retinol ws extrcted from serum or plsm nd quntified by reversed-phse HPLC by previously described procedures (17,18), except tht 2. ml of retinyl cette (finl concentrtion 6 g/l) ws used s the internl stndrd. Plsm trnsthyretin nd RBP. RBP nd trnsthyretin were mesured by using commercil RID pltes ccording to the mnufcturer s instructions. Quntifiction of RBP. To quntify free nd trnsthyretin-bound RBP, we used four independent methods: () extrction nd quntifiction of retinol from the HPLC peks; (b) quntifiction of REP nd trnsthyretin ctivity in HPLC peks by RID; (c) stndrdiztion with purified vitmin A-sturted free REP; nd (d) mesurement of ultrviolet bsorbncet 33 nm nd fluorescence t 418 nm (excittion wvelength, 33 nm). Vitmin A extrction from HPLC peks, nd nlysis. Multiple injections (e.g., six 2-pL smples) of norml humn plsm were chromtogrphed on the TK column s described bove, but with the concentrtion of sodium phosphte in the buffer decresed to 15 mmol/l. Ech chromtogrphic pek ws pooled, then concentrtedto 2 ul in Centricon 3 concentrtor (Amicon, Dnvers, MA) by room temperture centrifugtion in fixed-ngle rotor for 9 mm t 5 x g. RBP nd trnsthyretin concentrtions were determined in ech pool by RID. The vitmin A concentrtion in ech pek ws determined by reversedphse HPLC (14, 15). The mount of retinolextrcted from ech pek ws used to clculte the mount (nd percentge) of free nd trnsthyretin-bound REP in these smples. These clcultions were bsed on the fcts tht trnsthyretin forms n equimolr complex with REP, nd tht both free nd trnsthyretinbound RBP form n equimolr complex with retinol (1). Reltive moleculr msses used in these clcultions were 286, 22, nd 76, respectively for retinol,freerep, nd trnsthyretin-bound REP. tndrdiztion with purified RBP. Free REP derived from urine (98% pure) ws incubted for 18 ht 4#{176}C with, Tble 1. Properties of HPLC Peks from Norml Humn Plsm Eluted from TK 2 Column Pek ssignment Pek identifiction Retentiontime, mm Moleculrmss, D % of totl pek re % of totl extrctble vit. A in pek Excittion mx, nm Emissionmx, nm Immunologiclrectivity Anti-RBP Anti-trnsthyretin Figure 1. UpId- Trnsttiyretln- Free protein bound RBP RBP L I II Exclusion , 8.5, nd 35 molr excesses of retinol. Fluorescent intensities in ll the free REP liquotsincubted with retinol were substntilly greter thn in the liquot incubted without retinol. However, no significnt difference in fluorescent intensity ws observed between the liquots incubted with different molr excesses of retinol, indicting tht REP ws sturted with retinol in ll of these liquots. Vitmin A-sturted REP liquots were injected onto the TK column nd eluted under conditions identicl to those used for serum smples. All of the fluorescent mteril in the purified free REP liquots were eluted t the sme retentiontime s pek II. (Excess free retinol ppered to hve dsorbed onto the TK column mtrix.) The concentrtion of free REP in blood ws clculted by compring the pek,, re of pek in the serum smples with the pek res of different concentrtions of purified REP stndrd. Trnsthyretin-bound REP concentrtions were clculted by subtrcting these clculted free-rep concentrtions from their totl-rbp concentrtions (determined by RID). Quntifiction of RBP nd trnsthyretin by RID. Aliquots from ech TK pek were spotted on REP nd trnsthyretin RID pltes. We spotted 1 to 2 pl (5-1 times the recommended volume) onto ech plte to compenste for dilution of the smple during chromtogrphy. HPLC pek res from humn plsm or serum were correlted to REP nd trnsthyretin concentrtions mesured by RID. Lestsqures first-order liner regression nd correltion coeflicients were derived with igmplot (Jndel cientific, uslito CA) nd with A (ttisticl Anlysis ystem; A Institute, Cry, NC). Comprison of ultrviolet nd fluorescent pek res. Fluorescentnd ultrviolet intensity profiles of 5-pL liquot of norml humn or rt plsm were determined simultneously. We ssumed tht the ultrviolet bsorbnce t 33 mu reflected the mount of vitmin A in ech pek, wheres fluorescencews more specific for REP. Results nd Discussion Figure 1 illustrtes the spectrophotometric nd fluorescent spectr of typicl chromtogrm of humn plsm. Totl protein ws mesured spectrophotometriclly t 254 2$4.., UV UV 33.fl#{216}4I TIME 1.r.) Fig. 1. Typicl chromtogrmof 1 pl of fourfolddilutednorml + + humn plsm + - HPLCprocedures s descnbed in text Fluorescence nd bsofbnce (t 33 nm)were determined simultneously. Pek l trnthyretin-bound RBP;pekII, free RBP;pekL, hpoprotein-ssocited vitmin A CLINICALCHEMITRY, Vol. 35, No. 4,

3 m, but mesurement t 33 m ppered to be more specific for vitmin A or vitmin A-contining proteins in norml humn plsm. Free nd trnsthyretin-bound REP were esiest to detect fluorometriclly, becuse retinol ttched to free REP is highly fluorescent. Two peks hd immunologicl ctivity towrd REP ntibodies. Pek I, which ws eluted t 17.4 mm, ws identified s trnsthyretin-bound REP on the bsis of its moleculr mss, fluorescence chrcteristics, immunologicl ctivity to both nti- REP nd nti-trnsthyretin, nd vitmin A content, s shown in Tble 1. Pek I fluorescence vried linerly with injection volume (r =.9983) from 5 to 75 4 of plsm (Figure 2). Reproducibility ws good, the CV being 3.% for six replicte injections. Pek II, which ws eluted t 24.1 min, ws identified s free REP on the bsis of vitmin A content, immunologicl rectivity, nd moleculr mss. Pek II re vried linerly with plsm volume (r =.9998) from 5 to 754, the CV for six replicte smples being 3.3%. Pek II hs bout three times the re of pek I in the fluorescent chromtogrm of 25 ML of norml humn plsm, lthough this pek contins (t mot) sixth of the retinol in the smple. Pek II re lost its liner reltion to injection volume for volumes >75 4, nd becme inversely relted to smple volume t 3 4. This behvior suggests strong quenching t high REP concentrtions nd is consistent with the postulte tht free REP is much more fluorescent molecule thn is trnsthyretin-bound REP. Four independent methods were used to estimte the reltive mounts of free nd trnsthyretin-bound REP in norml humn serum: () extrction of retinol from the HPLC peks, (b) quntifiction by RID, (c) quntifictionof free REP by use of purified free REP, nd (d) comprison of ultrviolet nd fluorescent detection. All four methods gve similr estimtes of trnthyretin-bound REP concentrtions, with n verge CV between methods of 5.6%. Vitmin A extrction gve the lowest estimtes of trnsthyretinbound REP concentrtions, RID the highest. Agreement between estimtes for free REP concentrtions ws less good, with n verge CV of 16%. This ws not surprising, becuse free REP is present t lower concentrtion in plsm nd is locted in broder, more diffuse pek. Free REP concentrtions rnged from 2 to 12 mg/l nd trnsthyretin-bound REP concentrtionsrnged from 27 to 72 mg/l in these smples from norml persons. We do not know which of the four methods givesthe most nerly ccurte mesurements of free nd trnsthyretin- bound REP concentrtions in plsm. All four methods hve drwbcks tht could cuse inccurcies. Extrction of retinol from frctions represented by HPLC peks is both lbor intensive nd time consuming. Retinol-contining proteins could be lost during collection, concentrtion, storge, or ssy. RID requires concentrtion nd dilysis with potentil loss of retinol, or multiple spotting of smples with the resulting potentilly interfering buildup of slt. Compring ultrviolet nd fluorescent spectr is fst nd simple, nd mintins REP in more physiologicl environment, but is bsed on the ssumption tht ultrviolet detection gives n ccurte estimte of retinol, unffected by differences in moleculr configurtions or interfering substnces. Commercil REP sturted with retinol should give the best estimte of free REP concentrtion in plsm, but probbly does not. Unfortuntely, commercil preprtions of free REP re purified from humn urine, source tht contins vrible mounts of immunologiclly ctive REP molecules tht cnnot bind retinol (19), nd re therefore likely to be brekdown products. The mount of retinol tht cn be bound to these preprtions vries from lot to lot, indicting tht cution must be used when REP concentrtions re estimted by this method. Reportedly (2), lrge proportion of trnsthyretin-bound REP dissocites on TK 3 columns to form free REP. We did not see this problem with our TK 2 column. Trnsthyretin-bound REP did not dissocite on the TK 2 column. Indeed, no brekdown of trnsthyretin-bound REP or formtion of free REP ws observed when the frction corresponding to pek I ws collected nd rechromtogrphed three times. No lrgefreerep pek is detected by ultrviolet bsorbnce t 33m, or by RID. The rtio of free REP to totl REP (mesured by ultrviolet bsorbnce) does not chnge significntly when the concentrtion of totl REP is vried 15-fold, s would be expected if trnsthyretin-bound REP dissocited t low concentrtions of totl REP. The reltive percentges of free nd bound REP determined by our methods re consistent with previously reported results (6, 2). We believe tht differences in chromtogrphic technique, which resulted in poor seprtions of free nd trnsthyretin-bound REP on the TK 3 column, ccountfor their results. Figure 3 shows the correltion of free nd trnsthyretinbound REP plsm concentrtions (determined by verging the concentrtions estimted by the four methods used to quntitte REP) with totl REP determined by RID. The correltion with trnsthyretin-bound REP is negligibly bet- I Volume of Plsm lnj.ctsd. il- Fig. 2. Effect of plsm volume injected on RBP-continlng TK pek nes All determintionswere done in duplicte R.tlnel Binding Protein by RIO (mg / L) Fig. 3. CoiTeltion of RBP concentrtionsdetermined those determined by RID ANssysweredoneintriplicte by HPLC with 584 CLINICAL CHEMITRY, Vol. 35, No.4, 1989

4 ter (r =.865) thn the correltion with free REP (r =.846). Free REP, if linerly extrpolted, decreses to zero concentrtion t 19 mg of totl REP per liter. This suggests tht during vitmin A deficiency (concentrtion of REP in plsm <2 mg/l) little free RPB would be found in plsm. Most of the uncertinty in these correltions comes from the RID dt. We find dy-to-dy CV of 1.6% in REP s determined with RiD pltes, somewht higher CV thn the smple CV reported by the mnufcturer (8.5%). The dy-to-dy CV for HPLC pek II re ws 5.8%. The correltion of free nd trnsthyretin-bound REP with plsm retinol is shown in Figure 4. Agin, trnsthyretinbound REP correltes better (r =.699) with plsm retinol thn does free REP (r =.588). The correltion (Figure 5) between trnsthyretin-bound REP nd serum retinol is somewht higher thn the correltion between totl REP (determined by RID) nd plsm retinol (r =.575). The chromtogrms for serum nd plsm were very similr. The type of nticogulnt used (citrte or heprin) in the plsm smple hd no discernible effect (dt not shown). Pronounced hemolysis resulted in the ppernce of smll negtive pek centered t 16 mm. Hemolysis could potentilly interfere with this ssy, but did not in ny of our smples. The chromtogrphic proffle for rt plsm ws very similr to tht of humn serum or plsm (cf Figures 1 nd 6). Chrcteristic differences re tht the pek corresponding to high-moleculr-mss vitmin A (retention time 12.7 min) is more prominent in the rt, pek U (free REP) hs slightly longer retention time, nd shoulder on pek U is g35 3 C 25 C - Is #{149}.3 to (I o fr.. ROP #{163} trnithyrsdn-bound RBP r #{163} - - LA #{163}#{163},_. *- o A r-.355 o Plsm R.tlnol (smoi/l) Fig. 4. CorreltIonof RBP concentrtions determinedby HPLC with serum retinol concentrtions All ssyswere done In triplicte -j Plm Retinl (unei/l) Fig. 5. CorreltIonof plsm retinol with plsm retinol-binding proteins Totl RBP concentrtions determined by RID; plsm retinol determined s described In text. All ssys were done In duplicte I TIMEtmin) Fig.6. TypIcl chromtogrrn of 14 offourfolddiluted norml rt serum Peksidentified s in FIg. 1 much more frequently seen. We could detect no difference between free REP in the shoulder nd the min pek. REP peks in the rt were identified by their moleculr msses, their fluorescent properties, nd their similrity to humn chromtogrms. It is much esier to mke helthy rts vitmin A deficient thn it is to find otherwise nutritionlly dequte vitmin A deficient humns, so method tht cn be used to mesure REP in both species my become vluble reserch tool. This is simple method for ssying free nd trnthyretin-boundrep in serum or plsm of himirons nd rts. Its dvntges over the more commonly used immunologicl methods re tht it does not pper to be species specific, nd it cn seprtend mesure free nd trnsthyretinbound REP. This study ws supported by the United ttes Deprtment of Agriculture, Agriculturl Reserch ervice. Portions of this pper werepresented t the ntionl meeting of the AACC, n Frncisco, CA, July References 1. Goodmn D. The retunoids. Vol.2. New York: Acdemic Frees, 1984: Mute Y, mith JE, Much P, Goodmn D. Regultion of retunol-bunding protein metbolism by vitmin A sttus in the rt. J ) Biol Chem 1972;247: Mlli AK, mith JE, Goodmn D. Metbolism of retinolbinding protein nd vitmin A during hypervitminosis in the rt. J Lipid Ree 1975;16: mith FR, Goodmn 1)8. The effects of disese of the liver, thyroid nd kidneys on the trnsport of vitmin A in humn plsm. J Clin Invest 1971;5: Igenbleek V, Vn den chrieck HG, De Nyer P, De Visscher M. The role of retinol-binding protein in protein-clorie mlnutrition. Metbolism 1975;24: Fex G, Felding P. Fctors ffecting the concentrtion of free holo retinol-bindung protein in humn plsm. Eur J Clin Invest 1984;14: Peterson PA, Bergg#{227}rd I. Isoltion nd properties of humn retinol-trnsporting protein. J Biol Chem 1971;246: Beethm R, Dwny A, Lndon J, Cttell WR. A rdioimmunossy for retinol-binding protein in serum nd urine. Cliii Chem 1985;31: #{149} Prviinen NT, Ylitlo P. Immunoephelometric determintion of retinol-binding protein in serum nd urine. Clin Chem 1983;29: Bnkeon DD, Rifi N, ilvermn LM. Immunoturbidixnetric mesurement of serum retinol-bunding protein in renl nd heptic disese. Ann Cliii Biochem 1988;25: II CLINICAL CHEMITRY, Vol. 35, No. 4,

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