Radioimmunoassay of Somatomedin B

APPLICATION TO CLINICAL ANDPHYSIOLOGIC STUDIES

RosALYN S. YALow, KERSTIN HmL, and RoLF LugT

From the Solomon A. Berson Research Laboratory, Veterans AdministrationHospital, Bronx, NewYork 10468; the Department of Medicine, Mt. SinaiSchool of Medicine, The City University of NewYork, 10029; and theDepartment of Endocrinology and Metabolism, Karolinska Sjukhuset,Stockholm, Sweden

A B S T R A C T A radioimmunoassay has been developedfor Somatomedin B, a growth hormone-dependent factorthat stimulates DNAsynthesis in human glia-like cells.The sensitivity permits detection of this factor in humanplasma diluted 1:20,000 and in monkey plasma diluted1: 5,000. It is not measurable in nonprimate plasmadiluted 1: 20. The concentration in growth hormone-de-ficient adult patients is equivalent to 6.6±0.5 Ag/ml ofa highly purified somatomedin preparation. In acro-megaly the concentration is 19.3±2.3 tg/ml and fallsafter definitive therapy that results in a decrease inplasma growth hormone. In unextracted human plasmathe immunoreactive Somatomedin B is associated witha plasma protein at least as large as av-globulin and withan electrophoretic mobility on paper resembling thea-globulins. The level of Somatomedin B in the boundform in human plasma under steady-state conditionsmay depend on the rate of production of the peptide and/or the concentration of the plasma-binding protein. Atpresent there is no information concerning which ofthese is modulated by growth hormone. ImmunoreactiveSomatomedin B is found predominantly in Cohn plasmafractions III and IV, largely dissociated from theplasma-binding protein. The disappearance curves of la-beled purified Somatomedin B and of immunoreactiveSomatomedin B from acromegalic plasma administeredintravenously to a dog were superposable; the terminalportion of the disappearance curve having a half timeof almost an hour.

INTRODUCTIONThe existence of a pituitary hormone required for normalgrowth has been recognized since the beginning of this

Received for publication 24 July 1974 and in revisedform 30 August 1974.

century (1-3). However it was not until 1944 that theisolation and partial purification of a growth-stimulatingfactor from sheep pituitaries was effected (4). Growthhormone is unique among the peptide hormones in show-ing marked species specificity in its biologic action (5).The ineffectiveness in man of animal growth hormoneshas been shown to arise from distinct chemical differ-ences between the hormone from primates and fromlower species (6, 7). Marked species specificity is alsomanifest in the radioimmunoassay system, porcine andbovine growth hormones being less than 1/10,000 as im-munoreactive as primate growth hormone with the anti-human growth hormone antiserum prepared in ourlaboratory (8) and widely distributed by the HormoneDistribution Officer of the National Institute of Arthritisand Metabolic Diseases, National Institutes of Health,Bethesda, Md.

Before the development of radioimmunoassay metho-dology, it was not possible to measure growth hormonein normal human blood. However evidence had accumu-lated that the action of growth hormone on skeletal tis-sue is mediated by a substance whose concentration inplasma is growth hormone dependent. This factor wasoriginally called "sulfation factor" (SF)1 because itstimulates the uptake of radioactive sulfate in the costalcartilage of hypophysectomized rats (9). It has beenshown to decrease in human and rat plasma after hypo-physectomy and to increase after the administration ofhuman growth hormone to hypophysectomized patientsor beef growth hormone to hypophysectomized rats (9,10). Since the original reports (9, 10), the existence ofSF has been amply confirmed, and evidence has been

1Abbreviations used in this paper: GH, growth hormone;hGH, human growth hormone; NSILA, nonsuppressibleinsulin-like activity; SF, sulfation factor; Som B III, prepa-ration III.

The Journal of Clinical Investigation Volume 55 January 1975-127-137 127

accumulating suggesting additional properties for growthhormone (GH) -dependent serum factor(s). A moregeneral designation, "somatomedin," has been introduced(11), which is used as a generic term for any GH-de-pendent substance that appears to mediate the action ofGHor somatotropin. Although GHshows marked spe-cies specificity both with respect to its biologic and im-munologic properties, there has until now been no evi-dence for similar species specificity of any of the so-matomedins.

Recently at least three somatomedins with differencesin chemical and biologic properties have been described(12, 13); Somatomedin A which stimulates sulfate up-take by chick cartilage (14, 15); Somatomedin B whichstimulates DNAsynthesis in human glia-like cells (16);and Somatomedin C, a peptide more basic than somato-medin A or B and which stimulates sulfate uptake inrat cartilage (13). In the present report we describe thedevelopment of a radioimmunoassay for purified Somato-medin B and its application to some biochemical andclinical studies. The advantages of radioimmunoassay,namely its simplicity, sensitivity, and specificity, makefeasible the investigation of a host of chemical proce-dures and physiologic effects in numbers that would beprohibitive with the use of more tedious bioassays.

METHODS

RadioimmunoassayPreparation of antisera. Approximately 25 mg of KABI

luman purified Somatomedin B2 (preparation I) was pro-vided for immunization. Conjugation to guinea pig albumin(fraction V) was effected using the carbodiimide method ofGoodfriend, Levine, and Fasman (17) with only minorvariations. Six guinea pigs were immunized with the coupledpreparation containing 0.2-0.5 mg of Somatomedin B ho-mogenized in complete Freund's adjuvant according to theusual procedures of our laboratory (18). Antibody wasdetectable in the sera from all animals. However serumfrom only one animal was useful for radioimmunoassay atdilutions up to 1: 4,000 (antiserum 101) and from anotherat 1: 500 (antiserum 105). To conserve antigen, only thesetwo animals received more than four immunizing doses.The major portion of all bleedings of guinea pig 101 withsimilar titer was pooled and used for the studies reportedhere.

Preparation of radioiodine-labeled somatomedin: Somato-medin B preparations (I, II, and III) were labeled withradioiodine using the chloramine-T technique with onlyminor variations (18). Specific activity of the labeled prep-arations ranged from 10 to 50 ,ACi radioiodine/,g of mate-

2 Three different Somatomedin B preparations were ob-tained from AB KABI Research Department. PreparationI was purified from human plasma by Cohn fractionation,acid ethanol extraction, and gel chromatography (12).Preparation II was similar. Preparation III had been furtherpurified by electrophoresis and contained no SomatomedinA activity according to bioassay performed by Dr. KerstinHall.

rial. Generally '5I-labeled preparations were used for radio-immunoassay, and "3'I-labeled preparations were employedfor studies of the distribution and metabolism of somato-medin in dogs. Purification (18) of iodination mixtures waseffected either by Sephadex G-50 gel filtration or by starchgel electrophoresis using the method of Smithies (19).Generally, the eluates from starch gel were more suitablefor radioimmunoassay. For most studies the less pure prepa-ration II was used for labeling since contaminants wereremoved by the starch gel purification. More than 50% ofthe total labeled peptide could be bound in antibody excess.Using the best eluate from starch gel, generally more than90% was bindable to antibody. Thus approximately 40%of the labeled material that did not bind to antibody wasremoved by starch gel purification. No differences wereobserved between labeled Somatomedin B II or III afterpurification, and these preparations were used interchange-ably.

Standards. Preparation III (Som B III), which waspresumed to be the most highly purified Somatomedin Bpreparation made available to us, was used as standard,and all concentrations are expressed in terms of immuno-reactivity equivalent to a known weight of Som B III permilliliter.

Experimental conditions used for assay. The standarddiluent for the assay is 4% control guinea pig serum in 0.02M barbital buffer (pH 8.6). Antiserum 101 is used at afinal dilution up to 1: 4,000. 4-6 days of incubation at 40Care required to reach equilibrium at this dilution. Thevolume of the incubation mixture is 0.5 ml. Separation iseffected with 0.1 ml of uncoated charcoal (50 mg/ml 0.02-Mbarbital buffer). The double antibody method of separationof bound from free-labeled hormone can also be employed.To minimize the amount of second antibody required inthese assays the standard diluent has a lower concentrationof control guinea pig plasma, i.e., 1% control guinea pigplasma in the same barbital buffer.

Immunoreactive Somatomedin B in human plasma wasdetermined in a dilution of 1: 20,000 or greater and inmonkey plasma in a dilution of 1: 5,000 or greater. Plasmafrom other species including guinea pig, mouse, rat, cow,dog, sheep, pig, or rabbit was assayed at a 1: 20 dilution.Somatomedin B content of several of the plasma proteinfractions prepared by the Cohn method was also deter-mined. These fractions were a gift of the Protein Founda-tion, Boston, Mass. They were obtained as a dry powderand were soluble at a concentration of 1 mg/ml isotonicsaline. Preparations of Somatomedin A provided by KABI,of nonsuppressible insulin-like activity (NSILA) preparedby Dr. E. R. Froesch and provided to us by Dr. Jesse Rothand of Somatomedin C provided by Dr. Judson J. VanWyck were radioimmunoassayed for their Somatomedin Bcontent.

Fractionation procedures. All fractionation procedureswere carried out at 40 C. Before application to SephadexG-50 fine (1 X 50-cm) columns all samples including plasma,plasma protein fractions, or purified somatomedins werefortified with ['I]albumin and ['I] Na as marker mole-cules for void volume and salt peak, respectively. Whenfractionation was on Sephadex G-100 or G-200 columns['251]y-globulin, ['I]albumin, and ["'I]Na were used asmarkers. The zones of emergence of all components wereplotted as percent of elution volume between the markermolecules. Columns were equilibrated with eluting bufferbefore use; this was generally 1-4% control guinea pigserum in 0.02 M barbital buffer (pH 8.6).

128 R. S. Yalow, K. Hall, and R. Luft

Fractionation of endogenous hormone in human plasmaand of purified Som B III added to guinea pig plasma wascarried out on starch gel electrophoresis (6 V/cm for 18 h).After electrophoresis, sections were cut from the gel at 1-cm intervals, frozen for 3-4 h, thawed, and eluted with1%o control guinea pig plasma in 0.02 M barbital buffer.The gel particles were removed by centrifugation, and super-natant solutions were subjected to radioimmunoassay.

Endogenous human plasma somatomedin and labeled orunlabeled purified somatomedin added to guinea pig orhuman plasma were fractionated on paper electrophoresis(Whatman no. 3 paper, 0.1 M barbital buffer, pH 8.6, 6 V/cnm for 18 h). When labeled somatomedin was employed,the strip was heat-coagulated, autoradiographed, scannedfor radioactivity with a paper strip scanner, and thenstained to visualize the serum proteins. When unlabeledpurified or endogenous somatomedin was fractionated, thestrip was cut lengthwise. One-half of the strip was heatcoagulated and stained; the other half was sectioned at 1-cmintervals and eluted with standard diluent, and the eluateswere subjected to radioimmunoassay.

Rough estimates of the size of endogenous human plasmasomatomedin and purified Som B III added to guinea pigplasma were also obtained by ultracentrifugal analysis. Puri-fied Som B III was added to guinea pig plasma, equili-brated for 1 h, and then diluted 1: 5. Human plasma wasdiluted 1: 5. For comparison purposes [131I]human serumalbumin and ['1I]Jy-globulin in a 1: 5-dilution of humanplasma were also employed. 5 ml of each mixture wasadded in duplicate to tubes and sedimented in a Spincomodel L ultracentrifuge (Beckman Instruments, Inc., SpincoDiv., Palo Alto, Calif.) fitted with a bucket rotor (SW50.1) for 16 h at 40,000 rpm. After the centrifuge cameto rest, 0.5-ml samples were removed in succession fromthe top of each tube for assay of immunoreactivity or radio-activity. The concentration in each sample was expressed aspercent of the concentration in the uncentrifuged specimen.

Turnover studies. Turnover studies in dogs were per-formed by administering [l'I] Somatomedin B in a pulseinjection either alone or with 2-4 ml plasma from anacromegalic subj ect and which contained a high concen-tration of endogenous Somatomedin B activity. The labeledpreparation was administered with the immunoreactive soma-tomedin of plasma to monitor the accuracy and repro-ducibility of the injection procedure. Indwelling catheterswere inserted into the veins of two legs of the dog, onefor injection and the other for sampling. The catheter usedfor sampling was maintained patent with heparin. Heparin-ized blood samples were taken before and at regular in-tervals after injection. The blood was centrifuged imme-diately, and the supernatant plasma was stored frozenuntil assayed for radioactivity or somatomedin content.

Disappearance of labeled Somatomedin B and appearanceof radioiodide were analyzed by fractionation of plasmaradioactivity by hydrodynamic flow chromatoelectrophore-sis as well as by filtration on Sephadex G-50 columnsand by measuring the total radioactivity in the plasmasamples. In this way the fraction of the total radioactivitythat corresponded to intact labeled hormone was monitored.Disappearance of human plasma Somatomedin B after pulseinjection in a dog was determined by radioimmunoassay,and its form was determined by Sephadex gel filtration.

The rates of disappearance of labeled and unlabeled spe-cies were determined from a semilogarithmic plot of theplasma concentration as a function of time. To normalizevalues to become independent of the weight of the animal,

the plasma concentration was expressed as percent of theinjected dose per liter plasma, multiplied by the bodyweight of the dog.

Selection of patients. Human plasma samples assayedwere from male veteran patients referred to the Refer-ence Laboratory of the Bronx Veterans Administrationfor GH determinations. The diagnosis of under or oversecretion of GH was based on clinical findings and GHresponsiveness to standard glucose tolerance test for acro-megalic subjects and insulin tolerance tests for hypopitui-tary subjects. Normal subjects were those with normalGH responsiveness during the appropriate test. The samplesfrom male acromegalic subjects before and after definitivetherapy were supplied by Dr. Richard C. Dimond, Depart-ment of Endocrinology and Metabolism, Walter Reed ArmyInstitute of Research, Washington, D. C. All plasma sampleswere obtained in the fasting state in the morning.

RESULTS

To characterize the labeled somatomedin preparations,guinea pig plasma was added to the radioiodination mix-ture of each of the three somatomedin preparations ofdiffering purity (preparations I, IT, and III), and por-

PAPER

STARCHGEL ELECTROPHORESIS

-LB

wm100

'H -

0 50"0

5 -w

w

SEPHADEXG-50 GEL FILTRATKON

0 20 40 60 80 100PERCENTOF ELUTION VOLUME BETWEEN

VOID VOLUMEAND SALT PEAK

FIGURE 1 Analysis of iodination mixture of ['MI] Som BIII added to guinea pig plasma on chromatoelectrophoresis(top), starch gel electrophoresis (middle) and SephadexG-50 gel filtration (bottom). The major component of pro-tein-bound radioactivity appears as a single band in theprealbumin region on starch gel electrophoresis and has aKy -o 0.6 on Sephadex.

Radioimmunoassay of Somatomedin B 129

IODINATION MIXTURE-t25 I] Som B m

I ADDED TOh HUMAN

PLASMA

-) ADDED TO)t GUINEA PIG

9 PLASMA

0R I iN ANODE

FIGuRE 2 Analysis on paper electrophoresis of iodinationmixture of [2I1] Som B III. Autoradiographs are shownabove the corresponding stained paper strips for mixturesadded to human (top) and to guinea pig (bottom) plasmas.

tions of each were then fractionated by paper chromato-electrophoresis, starch gel electrophoresis, and SephadexG-50 gel filtration (Fig. 1). The patterns obtained withall three preparations were similar on paper chromato-electrophoresis and Sephadex gel fractionation. Onstarch gel the major peak of labeled Som B I was justslower than serum albumin, but for the other two prepa-rations the major peak was immediately in advance ofalbumin. On Sephadex G-50 gel filtration the major frac-tion of labeled Somatomedin B has a peak with a parti-tion coefficient, Kav, of about 0.6.

['I]Somatomedin B added to human or guinea pigplasma has an electrophoretic mobility on paper in theregion of the a-globulins (Fig. 2). This is in contrast toits mobility on starch gel where the same labeled prepa-ration migrates in the prealbumin region (Fig. 1). Theelectrophoretic mobility in the paper system is dependentonly on charge, and in the starch gel system, because ofmolecular sieving, it depends on both charge and size.The greater mobility on starch gel suggests that in thissystem there is no evidence for binding of labeled somato-medin to guinea pig serum proteins.

Using the radioimmunoassay system described in thesection on Methods, the smallest measurable concen-tration of immunoreactive Somatomedin B is ~ 25 pgSom B III/ml (Fig. 3). This sensitivity permits detec-tion of somatomedin in normal human plasma at dilutionsof 1: 20,000 or greater. No detectable immunoreactivecomponents were found in guinea pig, mouse, rat, cow,dog, sheep, pig, or rabbit plasmas at dilutions of 1: 20.Monkey plasma was assayed at a dilution of 1: 5,000.The concentration in normal monkey plasma is about2 Ag Som B III/ml. In 12 normal male subjects thefasting plasma somatomedin ranged from 4 to 20 ttgSom B IIII/ml with an average value of 9.8±1.5 AgSom B III/ml. The species specificity of the assay is

similar to that of the radioimmunoassay of human GHhormone, i.e., cross-reaction with primate hormone butnot with that from other animal species. [MI]Somato-medin B does not bind to antisera to insulin or GHwhenthe antisera are used in marked (100- to 1,000-fold) ex-cess. ['I] Somatomedin A or [PI] NSILA does notbind to the antiserum used in the immunoassay. No im-munoreactive Somatomedin B was detected in thepreparations of Somatomedin A, NSILA, or Somato-medin C that had been provided. Contamination of thesepreparations with as little as 1% Somatomedin B wouldhave been detected.

A scattergram of the immunoreactive somatomedincontent of fasting plasma from hypopituitary and acro-megalic subjects is shown in Fig. 4. There is some over-lap between the two groups. However the mean valuefor untreated acromegalic subjects (19.3±2.3 Ag Som BIII/ml) is about three times that of hypopituitary pa-tients (6.6±0.5 Ag Som B III/ml). In acromegalic pa-tients, definitive therapy (surgery or radiation) that re-sulted in a decrease in plasma GH concentration (Ta-ble I) also effected a reduction in the somatomedin con-tent of plasma.

Immunoreactive Somatomedin B in unextracted hu-man plasma elutes in the void volume on Sephadex G-50gel filtration and in the region of [JI]--v-globulin onSephadex G-100 and G-200 gel filtration (Fig. 5).Prior incubation of plasma diluted 1: 10 in 7 M ureafollowed by fractionation on a Sephadex G-50 columnequilibrated in and eluted with 7 Murea does not effect

1.0

GP 101|1:4,000 DILUTION OF ANTISERUM

mE 0.5-0

0.4 0.8 I.o 4.0SOMATOMEDINB CONCENTRATION

ng (Som B M)/mI

FIGURE 3 Standard curve for determination of Som B III.Standard diluent is 4% control guinea pig plasma in 0.02 Mbarbital buffer. Separation of bound and free ['2I]somato-medin was effected with uncoated charcoal.

130 R. S. Yalow, K. Hall, and R. Luft

dissociation of somatomedin immunoreactivity from theprotein-bound form.

The electrophoretic mobilities on paper of endogenoushuman plasma Somatomedin B and of purified Som BIII added to guinea pig plasma do not differ. The peak ofimmunoreactivity appears to be in the intra-a-globulinregion (Fig. 6a). However on starch gel electrophoresis,the major peak of endogenous human plasma somato-medin is close to the origin with some trailing up to thealbumin region whereas unlabeled purified SomatomedinB added to guinea pig plasma migrates in the prealbuminregion (Fig. 6b). Thus the labeled (Figs. 1 and 2) andunlabeled (Fig. 6) purified Somatomedin B in guineapig plasma behave indistinguishably on electrophoresis.

The similar electrophoretic mobilities on paper of en-dogenous plasma somatomedin and purified somatomedindo not permit decision as to whether or not purifiedsomatomedin binds to guinea pig plasma. Starch gel elec-trophoresis separates on the basis of size as well ascharge. Endogenous somatomedin is retarded becauseof its binding to a human serum protein. Purified So-matomedin B does not appear to bind to guinea pigplasma proteins and therefore migrates as a prealbumin,a mobility consistent with its charge as determinedfrom paper electrophoresis and its size as determined bySephadex gel filtration.

The sedimentation of endogenous Somatomedin B onultracentrifugation is consistent with its binding to aprotein approximating a-globulin in molecular weight(Fig. 7). In contrast the major fraction of purified un-labeled Somatomedin B does not bind to guinea pigplasma since its sedimentation velocity is less than thatof labeled albumin (Fig. 7) and resembles that of in-sulin (20). It should be noted that the pellet in the bot-tom 0.5 ml contained the remaining Somatomedin B andlabeled markers not found in the top nine portions shownin Fig. 7.

Somatomedin B in undiluted plasma is not detectablyadsorbed to charcoal. However when plasma is diluted

m 50-zh 40-

CE 30*

0-Co )0 20-

(M O' O

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ACROMEGALY

*:MEAN -....

0*

HYPOPITUITARISM

0MEAN

0 .-

FIGURE 4 Basal Somatomedin B concentrations in 22 adultmales with clinical acromegaly and proven hypersecretionof GH and in 17 adult males with diagnoses of chromo-phobe adenoma and GH deficiency proven by failure torespond to insulin-induced hypoglycemia.

TABLE IBasal Concentrations of GHand Somatomedin B

in Plasma of Acromegalic Subjectsbefore and after Therapy

hGH Somatomedin B

ng/ml p"g SOmBIII/mi

A. F. Pre 54 14.0Post 8 9.5

I. B. Pre 21 25.0Post 10 7.9

D. B. Pre 72 15.7Post 16 9.1

J. T. Pre 342 25.9Post 146 16.3

M. C. Pre 29 17.9Post

(a)ENDOGENOUSHUMANPLASMA

SOMATOMEDINB

I - G-O0itt AX~~G10

G-200

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40-cpm100

M3 I

6 20 40 60 80 100PERCENTOF ELUTION VOLUME BETWEEN

[125I]r-GLOBULIN AND 131I-(1)lLUH

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zz

0 -

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01)-

mz

5

LU

0E

0

Ct)

FIGURE 5 Gel filtration on Sephadex G-50, G-100, andG-200 of immunoreactive Somatomedin B in human plasma.On G-50, the [HI]albumin and [PI]-y-globulin markerssuperpose. Elution volume is expressed in terms of percentof volume between ['MI]1y-globulin and salt peak ('I-).

fied Somatomedin B before and immediately after in-travenous administration to a dog has a peak on Sepha-dex G-50 gel filtration with a Ka. - 0.6. Subsequently,when the intact hormone has disappeared, the void vol-ume component, corresponding presumably to the dam-aged labeled hormone, although initially a very minorcomponent, appears relatively more prominent and theradioiodide generated from degraded Somatomedin Bshows an absolute increase (Fig. 11). In spite of theapparent difference in size between endogenous humanSomatomedin B and radioiodinated Somatomedin B, asindicated by Sephadex gel filtration, their rates of disap-pearance after administration were superposable (Fig.12). For each form of somatomedin, the terminal por-tion of the disappearance curve has a half time of 50-60min.

30 1

I . ..

Som B I INGUINEA PIG PLASMA

2 13 1 4 5: '6 7 '8 9'10' 11'12'I .>

ORIGIN ANODE

( b )ALBUMIN Som B MALBUMIN IN GUINEA PIG

\s.~: PLASMA.- '"'

IL ..

T.O.NORMAL

W. S.15 - . HYPOPITUITARY

J. S.20 ::: HYPOPITUITARY

1. ,.

301

DISCUSSION

The term "somatomedin" is used to designate polypeptidefactors or substances in plasma that are, at least par-tially, GHdependent and appear to mediate the growth-promoting effect of GHin various tissues (21, 22). Theabsolute degree of purity of the Somatomedin B usedfor labeling and as a reference substance in our radio-immunoassay is not known. However, the observationthat the substance that is measured with the present as-say was approximately three times higher in serum inacromegaly than in hypopituitarism and consistently fellin the former group when there was a fall in plasma GHafter therapy, definitely speaks in favor of the measuredsubstance being a GH-dependent factor and thereforea somatomedin.

20 J. ~~~~~~~N.20 l ACROMEGALY

1 314'S'6'7'8 '9 '1O'I '12'ORIGIN ANODE

FIWURE 6 (a) Distribution on paper strip electrophoresisof endogenous Somatomedin B in human plasma (top)and Som B III added to guinea pig plasma (bottom). Aftercompletion of electrophoresis, the strip was air dried, cutdown the middle, and segments from half the strip eluted;the other half-strip was heat coagulated and stained. Thestained strip is shown directly below the pattern of im-munoreactivity. (b) Distribution of immunoreactivity onstarch gel electrophoresis of Som III added to guinea pigplasma (top) and of endogenous human plasma Somato-medin B (five bottom frames).

132 R. S. Yalow, K. Hall, and R. Luft

G-50ui

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z

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Z Ecr~

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~-0

H-

0IU)

L

C.L.ACROMEGALY

I

', .....

,,". i'.

.. :i,

The guinea pig antiserum developed in response to im-munization with human somatomedin B has a specificitydirected to a factor in human and monkey plasma thatwas not found in plasma from many nonprimate mam-malian species. The strong cross-reaction of monkeyplasma in the radioimmunoassay system rules out thepossibility that the antibody is directed against a plasmaprotein which is exclusively human. The observationthat the antiserum produced in response to immunizationwith Somatomedin B derived from human plasma reactsonly with primate plasma resembles the species specificityof antisera directed against human (h) GH. This spe-cies specificity is unique in antisera to peptide hormonesand raises the question as to whether human Somato-medin B might be a fragment of hGH. Failure of So-matomedin B to react with a antiserum to hGHdoes notrule out this possibility, since the antiserum may not bedirected toward that portion of the hGH molecule thatmight be somatomedin, or cleavage might so distort theconfiguration of the molecular fragment as to rule outthe possibility for interaction with antibody to the hGHmolecule.

SOMATOMEDINB LABELED MARKERS200 r-

z0I-<

00

Som B m ADDED TOZ 100- GUINEA PIG PLASMA [131] ALBUMINLi

0H | UNEXTRACTED'[125] SGLOBULINZ HUMANPLASMA:I

Luj0~

234'5'6 78 9 MO '2'3'45'6 7 8 9 1Th_TOP BOTTOM TOP BOTTOM

SUCCESSIVE 0.5-ml PORTIONS

FIGURE 7 Sedimentation of endogenous Somatomedin B inhuman plasma, of purified Som B III added to guinea pigplasma (left) and of labeled marker molecules in humanplasma (right). All plasmas were diluted 1: 5. After ultra-centrifugation of 5-ml samples for 16 h in a bucket rotor(SW 50.1) at 40,000 rpm, successive 0.5-ml samples wereremoved for radioimmunoassay of Somatomedin B contentor for assay of radioactivity content and were then com-pared to the mean Somatomedin B or radioactivity in un-centrifuged sample. The pellet contained in the bottom 0.5ml balanced the reduced concentrations of immunoreactivityor radioactivity of the upper portions.

TABLE I IImmunoreactive Somatomedin B in Cohn Fractions

of HumanPlasma

Fraction Concentration

ng (Som B III)/mg protein

I 65

II

STANDARDCURVE-COHNFRACTION I

o~~~~~~~ x

JIA~~~~~~~~~~~~~~~~

E0

C') 0.5-cm I

2.0 4.0SOMATOMEDINB CONCENTRATION

ng (Som B II)/mlr

FIGURE 8 Superposition of dilution curve of Cohn fractionson standard curve of SomB III.

In studies on the incorporation of labeled thymidineinto DNA of human glia-like cells, Somatomedin Bpurified from human plasma was used (16). Whetherif a similar fraction were purified from nonprimateplasma it would have been active in this system hasnot been studied. Recently it has demonstrated thatplasma from a variety of animal species stimulates sul-fate incoropration by human and monkey as well as pigrib cartilage (25). This suggests that SF does not havethe same biologic species specificity as does GH.

Somatomedin B activity in human plasma is knownto be associated with plasma proteins. In this study frac-tionation on Sephadex gel, starch gel, and ultracentri-fugation suggest that the molecular weight of the bind-ing protein is comparable to that of y-globulin, i.e., about150,000 daltons or greater. The electrophoretic mobilityof the binding protein corresponds to that of an a-globu-lin and does not differ from that of the purified unboundSomatomedin B.

We have been unable to demonstrate binding of la-beled or unlabeled Somatomedin B to guinea pig plasmaproteins. Because of the possibilities that the associationrate might be slow or dissociation rapid, we incubatedin whole guinea pig plasma for 8 h at room temperatureto speed association followed by 16 h at 4°C to retarddissociation. Nonetheless on starch gel electrophoresis,Sephahex gel filtration, or ultracentrifugal analysis, So-matomedin B behaved identically whether added before

fractionation to saline or normal guinea pig plasma. Asmall fraction of the labeled hormone, presumed to be adamaged moiety, binds nonspecifically to serum proteins,as is commonly observed with several damaged labeledpeptide hormones.

The spaces of distribution in the dog at 3 min afterintravenous administration of 'I-purified SomatomedinB or endogenous human somatomedin were similar andwere about twice plasma volume. The human plasma so-matomedin must therefore have dissociated, in part atleast, quite rapidly from its human binding protein afterthe dilution 250-500-fold in the blood of the dog. Be-cause the initial rates of disappearance of the somato-medins, labeled and unlabeled, were quite rapid (ti < 15min), their binding to dog plasma proteins could nothave been significant or their egress from the capillarybed would have been more retarded.

Som 5 IIc1,

FIl: ALB.I

I

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cpm

n

Cr)3LUD-J

LU-JLi(9

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(0

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z0

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0

a,

C/)LU

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z0

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C)

z0C-)z0Ld

0H

0U)

6 min POSTINJECTIONHUMANPLASMA

BEFORE INJECTION

cpm131I - 100

AI201 3 m POSTINJECTION

I

Al .1 = F210 -

n~iS

4-a

0

E0

8,000-

4,000

9 min POSTINJECTION

0 20 40 60 80 100PERCENT OF ELUTION VOLUME BETWEEN

[13'IIALBUMIN AND 131K-

F

FIGURE 10 Sephadex G-50 gel filtration of immunoreactivesomatomedin in human plasma (top) and in the plasmaof the dog 3 min (middle) and 9 min (bottom) after intra-venous administration of the human plasma. [11]albuminand [PI] Na were added to samples before application tothe columns to mark the void volume and salt peak re-spectively.

Consideration must be given as to whether the failureof unlabeled or labeled purified Somatomedin B to bindto serum proteins in the animal plasmas was due toalterations in structure attributable either to the initialextraction or subsequent iodination procedures. Whereasthis possibility cannot be ruled out, the failure of humanendogenous somatomedin, which dissociated rapidly dur-ing circulation in the dog, to rebind to dog plasma pro-teins cannot be accounted for by chemical alteration.The most likely explanation appears to be that the dogand guinea pig, at least, lack a specific binding proteinfor human Somatomedin B. However, we have also beenunable to demonstrate rebinding of labeled SomatomedinB to human plasma. Whether this is due to saturationof the human binding protein and a slow rate of ex-change or alteration of the labeled Somatomedin B is aproblem as yet unresolved. Hintz, Orsini, and VanCamp(26) have demonstrated dissociation of somatomedinactivity from a binding protein in human plasma duringSephadex gel filtration at an acid pH. The dissociatedmaterial reassociated at neutral pH with plasma proteinsthat had been freed of somatomedin.

The observation that the major fraction of immunore-active Somatomedin B in Cohn fractions III and IV isin the free form could be explained in three differentways: (a) Somatomedin B was altered by the fractiona-tions so that it could no longer bind to the binding pro-

2P000

12 min POSTINJECTION

I'*0 %

A A

I I~

150 min POSTINJECTION

_f __ ^- _- W

0 20 40 60 80 0ooPERCENTOF ELUTION VOLUMEBETWEEN

VOID VOLUMEAND SALT PEAK

FIGURE 11 Sephadex G-50 filtration patterns of radioac-tivity in dog plasma after intravenous administration of['I] Somatomedin B. The small peak in the void volumepresumably is altered labeled material bound nonspecificallyto serum protein; the central peak corresponds to intactlabeled somatomedin; the peak on the right is radioiodidereturned to the circulation after degradation of the [WI]-Somatomedin B.

10-8-

I-

4 A* [13ISOMATOMEDIN3: 4- .HUMAN PLASMA

1 N 9SOMATOMEDINB0

X 2-w

t 0.8

0 0.48

0.2-

0.2-1 . . . . . . . .30 60 90 120 150

TIME IN MINUTES

FIGURE 12 Superposable disappearance curves of intactpurified [I] Somatomedin B and immunoreactive Somato-medin B obtained from acromegalic plasma after intra-venous administration to a dog.

Radioimmunoassay of Somatomedin B 135

tein; (b) the binding protein was altered; (c) the bind-ing protein is in another Cohn fraction. The last is theleast likely since the binding protein appears to havethe characteristics of an a-globulin and hence shouldappear in fraction III and/or fraction IV. The failureto demonstrate rebinding of the labeled Somatomedin Bto unfractionated human plasma cannot be accounted forby alteration of the binding protein and is more con-sistent with explanation (a). If Somatomedin B ismodified during the extraction process so that it cannotrebind to the serum protein, what would be the effecton reactivity in the immune system employed in thisassay? The evidence is that bound and unbound Somato-medin B have almost equivalent immunoreactivity. Con-sider that total plasma protein is about 70 mg/ml, thatfractions III and IV account for about 25% of the totalprotein and that (from Table II) the mean concentrationof Somatomedin B (primarily in the unbound form) isabout 0.3 Ag/mg protein. The Somatomedin B concen-tration in unfractionated plasma should therefore beabout N (70 X 0.25 X 0.3) or 5 Ag/ml plasma. Thisagrees well with the mean concentration in normalplasma of 10 Ag/ml, allowing for losses in the Cohnfractionation.

It should be emphasized that the concentrations re-ported in the assay for Somatomedin B are in terms ofthe weight of preparation III standard. This standardis not necessarily completely pure. Nonetheless the ob-servation that the major fraction of the iodinated peptide,even before purification, binds in excess antiserum sug-gests that the assay is not based on a minor peptidecomponent in the preparation.

The level of Somatomedin B in the bound form in hu-man plasma under steady-state conditions may dependon the rate of production of the peptide and/or the con-centration of the plasma binding protein. At presentthere is no information concerning which of these ismodulated by GH.

ACKNOWLEDGMENTS

The authors are greatly indebted to Doctors Linda Fryk-lund, Hans Sievertsson, and Knut Uthne from the ResearchDepartment of AB KABI, Stockholm, who purified andsupplied to us the Somatomedin B preparations used forlabeling and for standards and to Mrs. Nancy Wu fortechnical assistance. The Cohn fractions of human plasmawere a gift of the Protein Foundation, Boston, Mass.

This is project no. 9678-01 of the Veterans Administrationand was also supported by grants from the Swedish Medi-cal Research Council (B74-19X-4224-01 and B 74-19X-34-11) and the Nordic Insulin Foundation.

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Radioimmunoassay of Somatomedin B 137