Radioimmunoassay of f3-microseminoprotein, a Prostatic-Secreted Protein Present in Sera of Both Men and Women

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1 LIN. HM. 35/7, (1989) Rdioimmunossy of f3-microseminoprotein, Prosttic-Secreted Protein Present in Ser of Both Men nd Women P.-A. Abrhmsson,1. Andersson,2 1. BjOrk, P. Fernlund,2 4H. LIIJ,2A. Murne,2 nd H. Welber3 We describe simple rdioimmunossy of /3-microseminoprotein, one of the three most bundnt secretory proteins of the prostte glnd. The detection limit of the ssy is 1 j.zg/l, nd its precision, expressed s the totl coefficient of vrition, is <1% for vlues between 1 nd 15 jzg/l. sing this ssy, we found tht /3-microseminoprotein immunorectivity ws present in ser from both sexes t bout the sme concentrtion. The protein detected hd the sme moleculr size on gel chromtogrphy s the protein isolted from seminl plsm, nd dilution curves for the ser prlleled tht for the pure protein. The findings suggest tht )3- microseminoprotein is present in serum of helthy subjects of both sexes nd tht it origintes in tissue other thn the prostte glnd. The rnge of the serum concentrtion ws -1.6 g/l (medin 4.1) for 51 helthy dult women nd g/l (medin 6.2) for 35 helthy dult men not older thn 4 yers. In mles with prosttic cncer the concentrtion in serum ws highly vrible nd often gretly incresed. The concentrtion of /3-microseminoprotein ws correlted with tht of cretinine in serum, suggesting tht the protein is eliminted-t lest prtly-from the circultion by glomerulr filtrtion. Little of the protein ws present in the urine of women. In urine from men the concentrtion ws high nd vrible, probbly becuse of locl contribution from the prostte glnd to the urethrl urine. AddItIonl Keyphrses: fertility. gondotropins 19Anhibin prosttic cncer reference vlues renl function seminl plsm urine The three most bundnt proteins in the secretions produced by the humn prostte glnd re prosttic cid phosphtse, prostte-specific ntigen, nd /3-microseminoprotein (1). All three proteins re present in seminl plsm t bout 1 gil ech (1-4). /3-Microseminoprotein, which hs n pprent moleculr mss of 14 to 16 kd on sodium dodecyl sulfte (SDS) electrophoresis (1, 5, 6), hs been clled /3-inhibin (7), f3-microseminoprotein (/3-MSP) (8), nd prosttic secretory protein of 94 mino cids (PSP94) (5) by vrious uthors. The mino cid sequence of /3-microseminoprotein hs been determined, both by protein sequencing methodology (6, 8, 9) nd by complementry DNA sequencing (1). A 94-mino cid peptide contining 1 cysteine residues nd five pirs of djcent bsic mino cids, nd lcking crbohydrte, /3-microseminoprotein shows little structurl similrity to other proteins. The biologicl function of J3-microseminoprotein is still Deprtments of rology, 2 linicl hemistry, nd Surgery, niversity of Lund, Generl Hospitl, Mlmo, Sweden. 4Address correspondence to this uthor t: Deprtment of linicl hemistry, Mlmo Generl Hospitl, S Mlmo, Sweden. Received Februry 28, 1989; cceptedapril 27, unknown. This protein ws originlly isolted from semen s fctor inhibiting the pituitry relese of follitropin (follicle-stimulting hormone) (5), but this ctivity hs now been questioned (11, 12) nd severl uthors hve rgued ginst the nme inhibin (13). The potentil of (3-microseminoprotein s mrker for prosttic disese (14-16) nd lso generl interest in its biologicl function hve prompted us to develop n ssy for this protein. Here we describe the properties of our rdioimmunossy of /3-microseminoprotein nd report the results of mesurements of this protein in serum nd urine of dult humns of both sexes. Mterils Mterils nd Methods The purifiction of /3-microseminoprotein from seminl plsm hs been described previously (1). The product obtined ws homogeneous, with n pprent moleculr mss of 16 kd s judged by SDS/polycrylmide gel electrophoresis, nd its mino cid composition nd NH2- terminl sequence were in greement with published dt (1, 5-1). We dissolved the purified protein in 5 mmol/l Ti-is H1 buffer, ph 7.4, contining Nl,.15 moljl (Ths sline), nd stored this stock solution in liquots t -2 #{176}. The concentrtion of /3-microseminoprotein in the stock solution ws determined by quntittive mino cid nlysis. Antiserum to /3-microseminoprotein ws obtined by immunizing New Zelnd White rbbits with the purified protein. The ntigen (.1 mg per niml) ws injected subcutneously on 1 different sites on the bck of ech niml. Freund s complete djuvnt ws used the first time nd Freund s incomplete djuvnt for repeted injections, done t monthly intervls. Antibody titers were checked regulrly, nd the ntiserum from the best-responding niml ws used. The ntiserum ws monospecific when tested by immunoelectrophoresis ginst norml seminl plsm. The single precipittion line obtined corresponded to the position for purified /3-microseminoprotein, but no precipittion line ws obtined with blood serum. Anti-rbbit IgG ntiserum ws rised in gots by stndrd methods (17). A sheep nti-rbbit immunoglobulin coupled to solid phse (Phrmci Decnting Suspension Ill) ws obtined from Phrmci, ppsl, Sweden. Purified mouse IgG ws kindly provided by Dr. Anders Grubb, Deprtment of linicl hemistry, Mlm#{246} Generl Hospitl, Mlm#{246}, Sweden. Bovine serum lbumin (ohn Frction V) ws obtined from Armour Phrmceuticl o. Ltd., stbourne,.k.; humn prolctmn from Hormone Lbortory, Aker Hospitl, Oslo, Norwy; polyethylene glycol (men Mr 6) from Kebo AB, Stockholm, Sweden; hlormine T from Merck AG, Drmstdt, F.R.G.; N1251 (17.4 kilg) from NN Products, Boston, MA; nd porcine insulin from Novo AS, openhgen, Denmrk. Blue Dextrn 2, moleculr-size mrker proteins for electrophoresis, Sephdex G-25 superfine, nd Superose 12 HR1O/3 LINIAL HMISTRY, Vol. 35, No. 7,

2 column were from Phrmci. Wter ws purified by n lg Spectrum R.O.1 system (The lg Group, Buckinghmhire,.K.). All other chemicls used were of nlyticl grde. Procedures Generl methods. SDSlpolycrylmide slb-gel electrophoresis ws performed in 1 to 15 g/l grdient gels (18) nd immunoelectrophoresis ws crried out with stndrd methodology (19). For mino cid nlysis we used Beckmn 63 utomtic mino cid nlyzer fter hydrolysis of n liquot of /3-microseminoprotein stock solution in n evcuted borosilicte glss tube for 24 h t 11#{176} in 6 mol/l H1, with norleucine dded s internl stndrd. Gel chromtogrphy. Gel chromtogrphy, s test of the homogeneity of lbeled f3-microseminoprotein nd for estimting the moleculr size of immunorecting mteril, ws performed t room temperture (pproximtely 2#{176}) on 1 x 31 mm column of Superose 12 HR 1/3. The elution buffer-sodium brbitl, 75 mmoljl, ph 8.6, contining 1. g of bovine serum lbumin nd.5 g of sodium side per liter-ws delivered with 215 HPL-pump (LKB, Bromm, Sweden) t flow rte of.7 ml/min. The column ws clibrted with Blue Dextrn 2 (verge moleculr mss 2. MD), mouse IgG (15 kd), bovine serum lbumin (68 kd), humn prolctin (23 kd), nd porcine insulin (6. kd). The effluent ws collected in 15-drop frctions nd the volume of ech frction ws determined by weighing. We monitored the frctions by rdioctivity mesurements when testing trcer homogeneity nd by rdioimmunossy in the size-estimtion experiments. Rdioiodintion. The iodintion ws performed t room temperture. To 11 x 55 mm glss tube with smll mgnetic stirrer we dded 5 L of N 251 (18.5 MBq) nd 5 L of purified f3-microseminoprotein (35 tg) in Trissline. The rection ws strted by dding 1 pl of hlormine T trthydrte dissolved in wter (2.5 g/l) nd stopped fter 3s by dding 1 L of sodium metbisulfite (5 gil) nd 3 L of 75 mmol/l sodium brbitl buffer, ph 8.6, contining 5 mg of sodium side per liter. The iodinted protein ws seprted from unrected iodide by chromtogrphy t 2#{176} on 1. x 1 cm column of Sephdex G-25 (superfine), equilibrted nd eluted with 75 mmoijl sodium brbitl buffer, ph 8.6, contining 5mg of sodium side per liter. The flow rte ws.7 ml/min nd the elute ws collected in tubes contining.5 ml of the elution buffer supplemented with 2 g of bovine serum lbumin per liter. Rdioimmunossy procedure. The ssy buffer ws 75 mmol/l sodium brbitl, ph 8.6, contining 2.5 g ech of disodium DTA nd bovine serum lbumin per liter. We used ntiserum diluted 4-fold in the ssy buffer. Serum from nonimmunized rbbits ws included to give concentrtion of rbbit serum in the working ntiserum solution corresponding to 16-fold dilution. Stndrds were prepred from the stock solution of purified /3-microseminoprotein by dilution in ssy buffer to give concentrtions rnging from to 35 pg/l. These 1498 LINIAL HMISTRY, Vol. 35, No. 7, 1989 were stored in liquots t -7 #{176}. We performed the ssy by mixing, in 11 x 55 mm polystyrene tubes, 1 L of stndrd or smple, 2.zL of diluted ntiserum, nd 2 L of 251-lbeled /3-microseminoprotein (diluted in ssy buffer to give bout 4 counts/mm per 2 ML). Nonspecific binding ws determined by excluding the ntiserum (i.e., using only serum from nonimmunized rbbits). After the mixture ws incubted t room temperture for 3 h, 1 L of pooled specimen of serum from norml humns, ll with /3-microseminoprotein <5 g/l, ws dded to the stndrd tubes nd to smple tubes with diluted serum or non-serum fluids. We dded 1 j.l of ssy buffer to the smple tubes with undiluted serum. Immeditely fterwrds, we precipitted immunoglobulins by dding 5 L of got ntirbbit IgG ntiserum diluted 4-fold in ssy buffer contining 5 g of polyethylene glycol per liter. Alterntively, we precipitted the bound trcer with.5 ml of suspension of solid-phse coupled sheep nti-rbbit immunoglobulin (Phrmci Decnting Suspension Ill). After letting the smples stnd for 1 h t room temperture, we centriftiged them t 2 x g nd 4 #{176} for 15 mm. The superntes were discrded nd the rdioctivity in the sediments ws mesured with 7% efficiency in n LKB-Wllc 1277 GmmMster 1-chnnel gmm counter (LKB, Bromm, Sweden). Smples were run in duplicte nd results were red from curve fitted to the counts obtined with the stndrds (ech point in triplicte) by using smoothed, cubic spline function with the stndrd errors of the mens s weighting fctors (2). Assy evlution. The sensitivity of the ssy ws defined s the reding on the stndrd curve t the men counts for the zero-concentrtion stndrd minus three stndrd devitions of the counts. We determined the intr-ssy coefficient of vrition (V) t three different levels by nlyzing three different smples 2 times in one ssy run, 1 in the beginning of the series nd 1 t the end. The intr-ssy V ws lso estimted from the duplicte vlues of smples (covering the rnge of vlues between 5 nd 175 pg/l) in 2 consecutive ssys. We estimted the totl V (intr-ssy + interssy) by ssying three different serum pools in 1 consecutive ssys. Aliquots of ech smple were kept t -2 #{176}, so tht repeted thwing ws voided. ch result ws the men of duplicte vlues. During the experimentl period two different trcer preprtions were used. We tested nlyticl recovery by dding three different mounts of purified /3-microseminoprotein from the stndrd stock solution to one serum smple from womn with low concentrtion of /3-microseminoprotein (5.5 ig/l) nd two smples from men, one with low concentrtion (9. u.gfl) nd the other with high (73.8 pg/l) concentrtion. The mounts dded were clculted to increse the concentrtion with 47.8, 87.5, nd 175 j.g/l, respectively. Assy of serum ntibodies to /3-microseminoprotein. This ws performed by n immunosorbent rdiossy ccording to ricsson et l. (21), /3-microseminoprotein lbeled s described bove being used s trcer. Serum cretinine. This ws determined in the routine clinicl chemistry lbortory with n lkline picric cid regent method (22). The norml reference intervls were 6-1.tmoIJL nd mol/l for dult women nd men, respectively. Gondotropins. Follitropin nd lutropmn (luteinizing hormone) were mesured by rdioimmunossy (23). Subjects nd ollection of Smples The reference popultion consisted of 51 helthy women

3 (ges 2-56 yers, 1 older thn 4) nd 43 helthy men (ges yers, eight older thn 4). Smples were lso obtined from 28 ptients (ges 56 to 98 yers, medin 74) with prosttic cncer. All ptients hd tumors in stges T3 or T4 of the Tumor, Node, nd Metstsis System of stging prostte cncers, which includes five stges of incresing severity (TO to T4) (24). Blood ws collected by venipuncture. We obtined serum by centrifligtion t 2 x g for 2 mm fter llowing the blood to clot for 6 mm t room temperture or overnight t 4#{176}. To obtin plsm, we smpled blood with DTA s nticogulnt. The serum nd plsm smples were stored t -2 #{176}. rine ws collected without dditives during 24 h from 24 helthy women (ges 2-56 yers) nd six helthy men (ges 2-4 yers). The volume ws mesured nd n liquot ws stored t -2 #{176} until nlyzed. Seminl plsm from helthy men ws obtined s described previously (1). It ws stored t -2 #{176}. Serum smples for correltion of serum cretinine with /3-microseminoprotein were selected from smples referred to the clinicl chemistry lbortory for routine tests. They were selected to give suitble distribution of serum cretinine vlues but with no regrd to the sex of the ptient, clinicl dt, or results of other blood tests. There were 46 smples from femles (ges yers, medin 65) nd 55 from mles (ges yers, medin 74). Results 3-Microseminoprotein Assy Lbeling of /3-microseminoprotein. With the lbeling procedure used, between 65% nd 85% of the rdioctivity (five lbeling experiments) ws incorported into the mteril eluting in the void volume of the G-25 column. Anlysis of this mteril by gel chromtogrphy on Superose 12 column, which hs higher resolving power thn the G-25 column, showed tht more thn 95% of the rdioctivity ws eluted in pek corresponding to the elution position of pure f3-microseminoprotein (determined in previous run on the sme column). The specific rdioctivity of the lbeled /3-microseminoprotein ws 42 GBq/g. The mount of trcer used in the ssy procedure (pproximtely 4 counts/mm) thus corresponded to bout 2 ng of lbeled /3-microseminoprotein per tube. Antiserum. Incubtion of the lbeled (3-microseminoprotein with vrious dilutions of the ntiserum, followed by precipittion of immunoglobulins with second ntibody, showed suitble dilution of the ntiserum in the incubtion mixture to be 1 -fold (Figure 1), i.e., 4-fold dilution in the working ntiserum solution. With ntiserum in excess-i.e., dilution <25-fold-more thn 9% of the trcer ws precipitted (Figure 1). When no ntiserum ws included (nonspecific binding), only few percent of the trcer ws precipitted (Figure 1). An eqully low nonspecific binding ws obtined when stndrds or smples (more thn 1 representtive smples tested) were included, mking correction for nonspecific binding in the ssy procedure unnecessry. Stndrd. The stndrds we used in our procedure were prepred in buffer contining bovine serum lbumin. Initilly we found tht the precipittion with second ntibody ws more reproducible with serum smples thn with stndrds. This phenomenon ws probbly cused by components (e.g., plsm proteins) present in serum smples, but not in the buffer used for the stndrds, tht mde the e e e I- V /25 1/1 1/41/16 no 1/5 1/2 1/8 ntiserum Antiserum dilution Fig. 1. Antiserum binding of 1251-lbeled /3-microseminoprotein Lbeled /3-microseminoprotein (pproximtely4 counts/mm,correspondingto 1 ng of lbeled ntigen) ws incubtedwith vrious dilutions of specific rbbitntiserum in totl volume of.5 ml t room temperture (pproximtely2#{176}). Theindicteddilution ofthentiserum referstothefinl dilution in the incubtionmixture,which lso continedserumfrom nonimmunizedrbbit, t 4-folddilution.After 3 h, immunoglobulin ws precipitted with second ntibody(see Mtenlsrid Methods). The rdioctivity ws mesuredin the precipitte obtined ftercentrifugtion (bound rdioctivity),ndthe resultws expressed s percentof totldded rdioctivity precipittes firmer nd more dhesive. The idel pproch would be to prepre the stndrd in serum free from /3-microseminoprotein, but we found it difficult to find such ser. Therefore, we dopted procedure in which 1 L of pooled serum with low concentrtion of /3-microseminoprotein ws dded to the stndrd tubes immeditely before the precipittion step. To mke the volumes equl in stndrds nd smples, the sme volume of buffer ws dded to the serum smple tubes. Seprting system. In the ssy procedure dopted for routine use we seprted bound nd free trcer by polyethylene glycol-reinforced second-ntibody precipittion. Seprtion with solid-phse-coupled nti-rbbit immunoglobulin (Phrmci Decnting Suspension Ill) gve identicl results (not shown) but ws not used becuse of the higher regent cost. Assy performnce. A typicl stndrd curve is shown in Figure 2 together with dilution curves for three different smples. The sensitivity of the ssy ws 1 p.g/l. stimtes of the intr-ssy V obtined with three different smples in single run were 3.3% (for smple with men concentrtion of 1.7 pgfl), 2.3% (smple men 81.7 tgfl), nd 2.4% (smple men 159 g/l), respectively. stimtion from smple duplictes in 2 different ssy runs gve 3.%. The estimtes of the totl V (i.e., intr-ssy plus inter-ssy) obtined for three different pooled ser in 1 consecutive ssys were 6.3% (t the men concentrtion of 9.8 g/l), 5.8% (t 8.2 g/l), nd 4.3% (t 156 g/l), respectively. Anlyticl recovery of dded purified /3-microseminoprotein vried between 94% nd 116% in the nine recovery experiments described under Mterils nd Methods. There ws no systemtic difference in recovery between the three test ser used for these experiments. Smpling. Vlues did not differ significntly between plsm nd serum (Student s pired two-tiled t-test) ccording to the results with plsm nd serum smples collected simultneously from 3 subjects. LINIAL HMISTRY, Vol. 35, No. 7,

4 Dilution fctor 5. Norml mle serum 1/256 1/64 1/16 1/4 1/1 1/128 1/32 1/8 1/2 fi -r.rr.,j S s,--. Norml femle serum --r S V S I- V 2 -J 4 2 JPmsttic cncer ptient serum Stndrd concentrtion (j.tgil) Fig. 2. Rdioimmunossystndrdcurve (#{149}) for /3-microseminoprotein nd dilution curves for serum from femle (), serum from ptientwith prosttic cncer (#{149}), nd seminlplsm(a) Smpleswere diluted in ssy buffer nd subjectedto the procedure with ddition of pooled serum t the end of the incubtion sdescribedin the text. The seminlplsmws prediluted 5-fold Stbility of /3-microseminoprotein in stored serum smples. Thirty-six serum smples (five from helthy men, five from helthy women, nd 26 from men with prosttic disese) tht hd been ssyed six months previously nd kept t -2 #{176} in the mentime were re-ssyed. The vlues obtined on the first occsion rnged from 5 to 74 g/l. The men difference between the second nd the first vlue ws plus 4.9%, with stndrd devition of 12.9%. /3-Microseminoprotein in Serum nd rine /3-Microseminoprotein ws detected not only in ser from men but lso in ser from women nd in urine. In Figure 2 re shown the dilution curves for the immunorectivity in serum from womn nd serum from mn, nd in seminl plsm, together with the stndrd curve obtined with purified /3-microseminoprotein. The curves re ll prllel. The immunorectivity in serum ws lso investigted by gel chromtogrphy, which seprtes molecules ccording to moleculr size. The results obtined with serum from norml mn, norml womn, nd ptient with prosttic cncer re shown in Figure 3, which, for comprison, lso shows the chromtogrms obtined with seminl plsm nd purified /3-microseminoprotein. In ll cses except for the prosttic-cncer ptient the immunorectivity ws eluted s single pek t position nd with width indistinguishble from tht of pure f3-microseminoprotein. According to the clibrtion of the column with moleculr-mss mrkers, the elution position corresponded to moleculr mss of bout 16 kd. In the ptient with prosttic cncer the min immunorectivity eluted s in the other smples but n extr minor immunorective component ws seen eluting before the min component (Figure 3). The molculr mss of the extr component ws estimted to exceed 15 kd. Possibly the two other serum smples contined similr component, but with concentrtion close to the detection limit of the ssy (Figure 3). Reference vlues. In the reference popultion, ser from mles tended to hve higher concentrtion of /3-microseminoprotein thn ser from femles, but the differ- ) Si ) p J\N r N Seminl plsm, , 4,4, 4,4, Pure 13-microseminoprotein i[ Frction number Fig. 3. Gel chromtogrphy of immunorective /3-microseminoprotein in serum smples nd seminl plsm Serum smples, seminl plsm, nd purified p-microseminoprotemn(.3 ml ech)were chromtogrphedon Superose12 column nd the effluent ws ssyed for /3-microseminoprotemn with the rdioimmunossyprocedure(with dditionof pooled serum t the end of the incubtion). The ser from the norml mlesnd femles were undiluted, serum from the prosttic-cncer ptientwsdilutedtwofold, nd the seminl plsm 5-fold;the concentrtionof the solutionof purified p-microseminoproteinws 187 igil. The elution positions for moleculr mss references (determined in seprte run) re indictedby numbered rrows:bluedextrn2. MD (1), mouselgg 15kD (2), bovine serum lbumin 68 kd (3), humn prolctmn23 kd (4), nd porcineinsulin 6. kd (5) ence ws not gret (Figure 4). If subjects older thn 4 yers were excluded, the difference between mles nd femles ws even less pronounced (Figure 4). The rnge ws -1.6 pgfl (medin 4.1) for the women nd gfl (medin 6.2) for the men not older thn 4 yers. There ws no correltion in the reference popultion between ge nd the concentrtion of (3-microseminoprotein in serum, either for women lone (r =.79), men lone (r =.224), or for ll subjects together (r =.154). The concentrtion of /3-microseminoprotein ws mesured in urine from 24 helthy women. Vlues were below the detection limit (1 jig/l) in 17 of the subjects, nd the 15 LINIALHMISTRY, Vol. 35, No. 7, 1989

5 1 5 ) 5) 2.,, I- 2 1 ( (S > V 5,. z S-13-Microseminoprotein (.Lg/L) Mles mrt S-13-Microseminoorotein (g/l1 Fig. 4. Distribution of vlues for concentrtion of /3-microseminoprotein in serum of helthy dults (51 women, 43 men) Shded brs represent results for men older thn 4 yers highest vlue mesured ws 7.7 tg/l. In urmnes from men the concentrtion ws much higher nd vrible. For six individuls the rnge ws 12.2 to 223 zgfl (medin 81.1 gfl). Prosttw-cncer ptients. The concentrtion of /3- microseminoprotein in ser from 28 ptients with prosttic cncer vried gretly. The rnge ws to 486 ug/l (medin 14.3 /Lg/L). Fourteen of the ptients hd vlues exceeding the upper reference limit nd four hd vlues below the lower limit. Ptients with more severe disese (stge T4) tended to hve higher vlues thn ptients in stge T3, difference tht ws sttisticlly significnt (P =.3) ccording to the Mnn-Whitney -test. Antibodies to /3-Microseminoprotein in Serum Ser from 1 helthy women, 1 helthy men (ll <4 yers old), nd 28 ptients with prosttic cncer were tested for the presence of circulting ntibodies to (3- microseminoprotein. None contined detectble ntibodies. /3-Microseminoprotein nd Serum retinine The reltion between the serum concentrtions of cretmine nd /3-microseminoprotein is shown in Figure 5 for 46 women nd 55 men. The low positive correltion between the two vribles (r =.49) is nevertheless sttisticlly significnt (P <.1). /3-Microseminoprotein nd Gondotropins Follitropin nd lutropin were mesured in the serum smples of the reference subjects. There ws no significnt correltion between the concentrtions of ny of the gondotropins on the one hnd nd /3-microseminoprotein on the other, either in women (n = 51) or men (n = 43). #{149}1 l I - I -- cb S-retlnlne (tmoi/l) Fig. 5. Reltion between concentrtions of f3-microseminoprotein nd cretinine in serum smples from 46 women () nd 55 men (#{149}) referred to the clinicl lbortory for routine clinicl chemistry tests The subjectswere selected only with regrd to the cretinine vlues, so s to include lrge rnge of cretinineconcentrtions.the equtionof the regressionline: y =.63x (r =.49, P <.1) Discussion /3-Microseminoprotein is present in seminl plsm in reltively high concentrtion. According to evidence obmined with histochemicl techniques (1, 14,25), northern blot nlysis (1), nd observtions in ptients lcking seminl vesicles (1) the /3-microseminoprotein in seminl plsm origintes from the prostte glnd. Not unexpectedly, therefore, /3-microseminoprotein my pper in serum of men with prosttic disese, s demonstrted with previously developed ssys for this protein (5, 26). Our results with the sensitive ssy described in this work show tht immunorective /3-microseminoprotein is present in serum of both men nd women nd t bout the sme concentrtion. Does this immunorectivity represent the sme protein tht hs been isolted from seminl plsm? Our belief tht this is so is supported by two of our findings: First, the immunorectivity in serum-both from men nd women-displyed dilution curve tht prlleled tht of the ssy stndrd curve, the ltter prepred with (3-microseminoprotein purified from seminl plsm. Second, frctiontion of the serum immunorectivity ccording to moleculr size showed tht lmost ll of it represents homogeneous component with the sme moleculr size s (3-microseminoprotein. Although only isoltion nd nlysis of the immunorective component could provide n ultimte proof, the evidence strongly suggests identity of the immunorectivity present in ser of women with /3-microseminoprotein. The nture of the minor immunorective component with lrger moleculr size thn (3-microseminoprotein present in the smple from the ptient with prosttic cncer-nd perhps lso in ser of helthy subjects-is uncler. The component could be n ggregted form of /3-microseminoprotein or perhps f3-microseminoprotein in complex with high-moleculr-mss binder in the ptient s plsm. The concentrtion of /3-microseminoprotein in ser of women, when mesured with our ssy, is lmost the sme s in helthy men not older thn 4 yers, n ge below which benign prosttic hyperplsi or prosttic cncer is rre. Although the men vlue for men is sttisticlly higher thn for women, the overlp is lrge, nd one my conclude tht, for helthy subjects, the concentrtion is essentilly the sme in both sexes. This finding suggests tht the prostte glnd is not n importnt source for LINIALHMISTRY, Vol. 35, No. 7,

6 serum /3-microseminoprotein in helthy men, nd tht blood of men nd women hve common source for this protein. In men with prosttic cncer the concentrtion of (3- microseminoprotein in serum ws often mrkedly incresed, similr to wht hs been observed with other prostte-specific mrkers, such s cid phosphtse nd prostte-specific ntigen. This increse most probbly represents direct relese of /3-microseminoprotein into blood from the disesed prostte glnd, nd it my well be tht the high vlues occsionlly seen in subjectively helthy men older thn 4 yers lso represent contributions from the prostte glnd. The relese of /3-microseminoprotein into the circultion s consequence of disese process ffecting the tissue producing this protein might stimulte the production of ntibodies directed ginst (3-microseminoprotein. If present, such ntibodies would interfere with our ssy nd probbly cuse flsely incresed vlues. Our filure to detect ntibodies in ny of our group of ptients with prosttic disese (mny with high vlues for /3-microseminoprotein in their blood) suggests tht this does not hppen frequently. Although it ppers to be somewht lrger in SDS electrophoresis nd gel chromtogrphy (16 kd), (3-microseminoprotein is smll protein of 94 mino cids with clculted moleculr mss of bout 11 kd (1,5,6,8-1). Proteins of this size re usully more or less freely filtered in the renl glomeruli. We therefore mesured /3-microseminoprotein in urine from femles nd from mles s well. In femles the concentrtion ws low, more thn hlf of the ptients hving vlues below the detection limit of our ssy. Men hd much higher nd more vrible concentrtions of /3-microseminoprotein in their urine, nd occsionlly very high vlues were encountered. The low vlues in women suggest tht (3-microseminoprotein, if filtered in the glomeruli, is effectively rebsorbed by the tubule cells nd tht the urine finlly is lmost free of the protein. The high vlues seen for urine from men thus probbly represent locl contribution from the prostte glnd to the urethrl urine. Filtering of /3-microseminoprotein in the renl glomeruli nd subsequent rebsorption in the tubuli might be mjor ctbolic route for this protein. One would then expect correltion between the concentrtions in blood of (3-microseminoprotein nd mrker of glomerulr ifirtion rte, such s cretinine. Our mesurements of f3-microseminoprotein in number of ptients with vrious serum cretinine concentrtions showed sttisticlly significnt positive correltion between these two vribles, giving support to the hypothesis tht t lest prt of the ctbolism of /3-microseminoprotein tkes plce by glomerulr filtrtion. If the input of /3-microseminoprotein into the circultion vried little in these ptients nd glomerulr filtrtion ws responsible for the elimintion of the protein, one would hve expected correltion higher thn tht observed. But mny of the ptients in this study hd severe diseses besides their renl insufficiency, nd it is not unlikely tht mny of the high vlues for (3-microseminoprotein seen in these ptients were prtly cused by n incresed input of (3-microseminoprotein into the circultion s consequence of disese ffecting n orgn synthesizing (3-microseminoprotein, e.g., the prostte glnd in the men. The function of (3-microseminoprotein is still unknown. rlier proposls (7) tht /3-microseminoprotein hs inhibin ctivity-i.e., inhibition of relese of follitropin from the pituitry-hve been questioned (11, 12). This is consistent with our finding of zero correltion between /3-microseminoprotein nd follitropin. This work ws supported by grnts from the Swedish Medicl Reserch ouncil (Project No. B88-3X-5913 nd B88-13X-793), the Fculty of Medicine t the niversity of Lund, the Albert PMilsson Foundtion, the Gret nd John Kock Foundtion, the John nd August Persson Foundtion, the Ann Lis nd Sven- rik Lundgren Foundtion, nd the ncer Reserch Fund t MlmO Generl Hospitl. References 1. Lilj H, Abrhmsson P-A. Three predominnt proteins socreted by the humn prostte glnd. Prostte 1988;12: Vubourdolle M, lvel JP, Gonzles J, Glli A. vlution of cidphosphtse isoenzymes in seminl fluid from normozoospermic, oligozoospermic, zoospermic nd sthenotertozoospermic men. Andrologi 1985;17: Wng M, Kuriym M, PpsideroLD, et l. Prostte ntigen of humn cncer ptients. Methods ncer Res 1982;19: Tsud R, Inoue T, Hr M. A seminl plsm specific ntigen of prostte glnd. Jpn J Leg Med 1982;36: Dub#{233} JY, Frenette G, Pquin R, et l. Isoltion from humn seminl plsm of n bundnt 16 kd protein originting from the prostte, its identifiction with 94-residue peptide originlly described s /3-inhibin. J Androl 1987;8: Seidh NG, Arbtti NJ, Rochemont J, Sheth AR, hr#{233}tien M. omplete mino cid sequence of humn seminl plsm /3- inhibin. FBS Lett 1984;175: Thkur AN, Vze AY, Dtttreynurty B, Arbtti NH, Sheth AR. Isoltion nd chrcteriztion of inhibin from humn seminl plsm. Indin J xp Biol 1978;16: Akiym K, Yoshiok Y, Schmid K, et l. The mino cid sequence of humn /3-microseminoprotein. Biochim Biophys Acts 1985;829: Johnsson J, Sheth A, ederlund, Jornvll H. Anlysis of n inhibin preprtion revels pprent identity between peptide with inhibin-like ctivity nd sperm-coting ntigen. FBS Lett 1984;176: Mbiky M, Nolet S, Fournier S, et!. Moleculr cloning nd sequence of the edna for 94-mino cid seminl plsm protein secreted by the humn prostte. DNA 1987;6: Kohn S, Froys B, ederlund, et l. Lck of in vitro inhibin ctivity of 94-residue peptide isolted from humn seminl plsm, nd of synthetic replicte of its -terminl 28-residue segment. FBS Lett 1986;199: Gordon WL, Liu WK, Akiym K, et!. /3-Microseminoprotein (/3-MSP)is not n inhibm. Biol Reprod 1987;36: Burger HG, Igrshi M. Inhibin: definition nd nomenclture, including relted substnces [Letter]. J lin ndocrinol Metb 1988;66: Dube JY, Pelletier G, Ggnon P, Trembly RR. Immunohistochemicl locliztion of prosttic secretory protein of 94 mino cids in norml prosttic tissue, in primry prosttic tumors nd in their metstses. J rol 1987;138: Trembly J, Frenette G, Trembly RR, Dupont A, Thbet M, Dub#{233} JY. xcretion of three mjor prosttic secretory proteins in the urine of norml men nd ptients with benign prosttic hypertrophy or prostte cncer. Prostte 1987;1: Abrhmsson P-A, Lilj H, Flkmer S, WdstrOm LB. Immunohitochemicl distribution of the three predominnt secretory proteins in the prenchym of hyperplstic nd neoplstic prostte glnds. Prostte 1988;12: Thorell JI, Lrson SM. Rdioimmunossy nd relted techniques. Methodology nd clinicl pplictions. Sint Louis, MO: V Mosby, 1978: Blobel, Doberstein B. Trnsfer of proteins cross membrnes. I. Presence of proteolyticlly processed nd unprocessed nscent immunoglobulin light chins on membrne-bound ribosomes of murine myelom. J ell Biol 1975;67: Scheidegger JJ. ne micro-m#{233}thode l imznuno#{232}lectrophor#{233}se. mt Arch Allergy 1955;7: LINIAL HMISTRY, Vol. 35, No. 7, 1989

7 2. Reinsch H. Smoothing by plme functions. Numerische Mtherntik 1967;1O: ricsson -B, Lrsson I, Murne A, Thorell JL A new sensitive iznmunosorbent rdiossy forthe detection ofcirculting ntibodies to polypeptide hormones nd proteins. Scnd J lin Lb Inve8t 1984;44: Brtel H, BOhmer M. ine Mikrometode zur Kretininbestimmung. lin him Acts 1971;32: Thorell JI, HolmstrOm B. Production highly purified humn follicle-stimulting of ntiser ginst hormone, luteinizing hormone nd thyroid-stimulting hormone. J ndocrinol 1976;7: Wllce DM, hisholm GD, Hendry WF. TNM clssifiction for urologicl tumors (I) Br J rol 1975;47: Doctor VM, Sheth AR, Simh MM, Arbtti NJ, Averi JP, Sheth NA. Studiesonimmunocytochemicl locliztion ofinhibinlike mteril in humn prosttic tissue: comprison of its distribution in norml, benign nd mlignnt prostte. Br J ncer 1986;53: Sthe VS, Sheth NA, Phdke MA, Sheth AR, Zveri YP. Biosynthesis nd locliztion of inhibin in humn prostte. Prostte 1987;1O: LINIAL HMISTRY, Vol. 35, No. 7,