The electrolytic preparation of bioactive radioiodinated parathyroid hormone of high specific activity

AN.41

yTI(

A,

HIO(‘,,EMISI

67-73

RY 92.

The Electrolytic

( 19791

Preparation of Bioactive Radioiodinated Hormone of High Specific Activity’

SUSAN T.

PA.UI A AND WII

NIELSEN.~

Q.

BARRETT.:’ MARGARETI W. F. NFUMAN“

A technique

for

labeled

the

with

preparation

June

of microgram

carrier-free

‘“‘1

to a specific

activity

of bovine of

1300

parathyroid Ci/‘mmol

hormone

is described.

A

potential and the

the reaction. and KCI solution

bPTH elutes in 3 11 guanidine

a voltmeter to monitor the (via a calomel electrode)

as a silngle HCI-2.3

species when chromatographed ~VIformic acid. and it retains

that bPTH potency

labeled

paper

ia hased

on

work

supported

hy

NIH

Grants AM-17074 and IF39DE-051 I9 and in part by DOE Contract EY76-C02-3490 at the University of RoChester and ha\ heen a\Ggned Report No. UK-3490-

America\. ,’ Present University

quantities

hattery to drive reference-saturated

To be useful for hormone metabolism or receptor interaction studies, labeled parathyroid hormone must be detectable at picomolar concentrations. Therefore. in addition to retaining biologic activity after labeling. the hormone must also be labeled to a specific activity of at least 10001Ci/mmol. An electrolytic iodination procedure has been described t 1) which produced biologically active bovine parathyroid hormone

ICI

1978

iodination. making it feasible setup requires only a small to a saturated KCI solution. a

hioassayed i,r ~i~.,j. It is evident \uffcr in regard to its biological

1217. ” Present

29.

restructured and simplified apparatus was used for electrolytic to use reaction volumes of 1001 to 200 ml. The miniaturized platinum crucible connected via an agar-KCI salt bridge

[““I]-labeled equilibrated

’ ‘fhi\

NFUMAN.

1.1~~

Received

(bPTH)

Parathyroid

address:

Biomedical

Inc.. Wilmington. address: Department School of Medicine.

Reswrch Del. of Neu

Laboratory, 19897. Medicine. Haven.

065 IO. I Reprint requests should be addressed William F. Neuman. Department of Radialion and Biophysics. University of Roche\l.er Center. Rochester. N. Y. 14641.

Yale Corm. to: Dr. Biology Medical

to ri high

specilic

full

difference platinum

between crucible.

on a Biogel biologic

P- 10 column activity when

activity

with

“‘I

does

the The

not

tbPTHY’ which was free from detectable damage at specific activities of -100 Ciimmole. Unfortunately. as described, this technique is unsuitable for the preparation of high specific activity labeled bPTH. Although other methods. such as the chloramine-T method. can be used to produce high specific activity iodinated bPTH. the labeled hormone is no longer biologically active and may suffer some fragmentation as well (2,3). The use of lactoperoxidase for labeling bPTH has been reported (4,51. Unfortunately. complications in interpreting these studies arise because the location of the label within the hormone molecule has not been demonstrated. Further, because a bPTH preparation of low biologic activity was used, the possibility cannot be excluded that biologically inactive hormone molecules might be preferentially labeled. ’ Abbreviation\ used: bPTH. bovlnr parathyroid hormone: BSA. hovine \erumalbumin: TCA. trichlorowetis: acid: CMC. wdium carbouymethyl cc’llulc~w.

68

NIEI.SEN

Because of these considerations, the electrolytic iodination apparatus was modified such that microgram quantities of the hormone could be readily labeled. The constraints imposed by pH, voltage. and hormone concentrations were investigated. Under optimum conditions, the preparation of high specific activity hormone (== 1300 Ciimmol) is possible. while the absolute amount of ““1 necessary can be held to a reasonable level ( 10 to IS mCi). It was of paramount importance. however. to establish that the iodinated hormone was not damaged by the intense radioactivity. per se, as has been suggested (3). Its full biologic activity. demonstrated ill \vi\vo using the chick hypercalcemic bioassay of Parsons rt trl. (6) is reported here. With the availability of iodinated parathyroid hormone at this specific activity. it is now possible to approximate physiological hormone levels in radiotracer experiments. using nanogram quantities or less. MATERIALS

AND METHODS

Initial attempts to scale down the originally described ( I) controlled voltage electrolysis procedure for iodination were hampered by the rather clumsy membrane cathode. The simultaneous use of the KCI-agar bridge as a voltage-sensing electrode as well as a current-delivering cathode satisfied the need to separate the oxidation and reduction reaction, yet eliminated the bulky membrane cathode. In addition, as a precautionary measure against radiation exposure or possible airborn losses of lz512 during iodinations, the electrolysis anode vessel (a small platinum dish of 0.3-0.5 ml capacity) was enclosed in a lead shield modified from a commercial isotope shipment container. The entire anode assembly was surrounded by an ice bath and the reaction mixture was stirred magnetically during the iodination process. The agar-KCI salt bridges were freshly prepared for each iodination using a I%

k’/ A/

agar-saturated KCI solution and a IO-in. length of polyethylene tubing (0.08s in. i.d. ). Smaller diameter tubing produced unsatisfactory bridges. Preceding each iodination, the pH was measured with a microelectrode and adjusted if necessary. During the iodination procedure, a constant current of 40 PA was maintained until the maximum voltage was reached, 750 mV unless noted otherwise. The current. theoretically, is directly proportional to the rate and amount of iodide oxidized to iodine. However, in practical terms, this relation is only approximate at the nanomolar levels involved. In general, the current of 40 PA lasted from I to 5 min and baseline current (salt conductance) was reached in from 5 to I5 min. Accordingly, iodinations were usually terminated after IS to 20 min of electrolysis. 30 min maximum. A diagrammatic representation of the modified iodination apparatus is shown in Fig. 1. In a typical iodination reaction the following reagents were added: 100 ~1 of NaI (4.5 x lo-” mol, containing lznI-) in 0.1 N NaOH, 43 Fg of bovine parathyroid hormone (4.5 x IO-” mol), dissolved in lop.1 of 1.0 N HCI. 25 ~1 of H,O, and I5 ~1 of 0.25 M sodium phosphate buffer, containing I.0 M NaCI, pH 7.4. Following the conclusion of the electrolytic process, any I2 remaining was reduced to I- by the addition of 5 ~1 of mercaptoethanol solution (60 pmol). The reaction mixture was acidified with HCI and 250 Fg of bovine serum albumin (BSA) was added to reduce adsorptive losses: unreacted lz31 was separated on a Biogel P-2 column. The eluant was 0.01 N HCI containing BSA, 2 mgiml. Following the iodination. the percentage of radioactivity incorporated into the hormone was determined in some experiments by precipitation of the reaction mixture in IO% cold trichloroacetic acid using BSA as carrier protein, and in some experiments by Biogel P-10 gel filtration chromatography (see Fig. 3). Since in all cases. the ratio of iodide to hormone was maintained at unity the percentage labeled

PREPARATION

OF

69

[““I]IQDQ-hPTH

MICRO-MAGNETIC STIRRING

SAT

FIN,.

I. Schematic

jl~~l]iudo-bPTH. hath

during

KC,

representation

Reaction the

iodination

BAR

vulume\ procedure.

of iodlrnation

apparatus

suitable

for

of 100 to 200 pl are used. The closed Two

1.5-V

batteries

represents the specific activity of the hormone. i.e., as a percentage of the total disintegrations per minute added to the reaction mixture. (In Fig. 2, for example. the fraction of lzr,I incorporated into the hormone is determined by dividing the counts per minute under the peak. of intact

IDE

in series

and

the

preparation

lead container two

500.ohm

of high

specific

is wrrounded variable

resistors

activity

by an ice are

used.

bPTH by the total counts per minute layered onto the column. Recoveries of radioactivity from the columns were greater than 98%). The two methods gave similar results. Radioactive samples were counted in a Beckman Biogamma counter. c’ilich h~pc~c~trl~~c~nli~~hiorrs.,rry. Six-day-

C

1

40

80 FRACTION

FIG. 2. Biogel P-10 gel filtration analy& of [r”‘I]iodo-bPTH on a column of Biogel P-2, an aliquot of the [‘9liodo-bPfH guanidine-2.3 M formic acid containing column markers

120

NUMBER

prepared electrolytically. Following to be used in the chick bioassay was added and layered onto a Biogel P-IO column.

deiodination to 1 ml of 3 M I.5 Y 70 cm.

which had been previously equilibrated with the guanidine-formic acid eluant. One-milliliter fractions were collected at a flow rate of 15 to 20 ml/h. DB. C. and T denote the elution positions of dextran blue 2000 (void volume marher). cytochrome (‘, and tetracycline (included volume marker). As in almost every sample of hormone tested, there is a small amount of higher molecular weight contaminant rseen preceding the main hormone peak. If desired this can be removed by adsorption of the hormone (and elution) using silica (Quso).

70

NIEISEN ‘TABLE

PH 6.4 7.4 8.4

RESULTS

I

Percentage iodinatic>n 2 SEM 56.73 56.60 59.33

k7 Al.

+ 2.94 -t 5.10 i- 4.13

II 3 3 3

old chicks weighing 43 to 52 g were injected intravenously with 0.3 ml of acetate-buffered vehicle, pH 4.0, as described by Parsons c>t trl. (6), containing varying amounts of unlabeled bPTH or [rZsI]iodobPTH. Both the radioactively labeled and the unlabeled bPTH originated from the same bPTH sample, in order to eliminate any sample-to-sample variation. Control chicks were injected with vehicle alone. At least five birds were included in each dose group. One hour later. the chicks were sacrificed by decapitation. Blood was collected and allowed to clot. Equal volumes of serum and 10% TCA were mixed, after which the samples were kept on ice for 1 h and then centrifuged. The supernatant was analyzed for Ca?+ fluorometrically by EDTA titration according to the method of Borle (7). The data were analyzed using programs Stat I-05A (sum for two means) from the Hewlett Packard 65 programmable calculator Stat Pat 1. A4crtc~rirrl.s. A Beckman digital pH meter, Mode1 3500. was employed as the voltmeter. Selected lots of highly purified bovine parathyroid hormone (1000-1400 Uimg) were purchased from Inolex, Chicago, and CMCpurified bPTH (2500-4000 Uimg) was the generous gift of Dr. H. Keutman. Na”“I was purchased from New England Nuclear, Boston, and used within 1week of purchase. One-day-old male chicks of the Rhode Island Red x Barred Plymouth Rock strain were purchased from Arbor Acres Farm, Glassenbury, Connecticut. Biogel P-2 and Biogel P-10 were from Bio-Rad Laboratories, Rockville Center, New York.

As shown in Table I, variation of the pH of the reaction medium from 6.4 to 8.4 did not alter iodination efficiency. In all cases. 55 to 60%’ of the added label was incorporated into bPTH (Table I). Moreover, no detectable alteration in the molecular weight of the labeled product as the result of pH variation was revealed by gel filtration chromatography. Analysis of the species iodinated at pH 6.4 showed two peaks of radioactivity. The first was a single symmetrical peak that eluted just after cytochrome c (MW 12.400). This peak had a distribution coefficient of 0.18, which corresponds on this chromatographic system to a molecular weight of 9500 (8). No secondary peaks or shoulders. suggestive of damage to the hormone, were observed. The peak of radioactivity eluting after tetracycline represents unreacted iodide. Gel filtration analysis of samples iodinated at pH 7.4 or 8.4 produced essentially identical radioactivity profiles (data not shown). In contrast, alteration of the maximum voltage did affect iodide incorporation. As shown in Table 2, when the potential difference generated did not exceed 580 mV, only 37 2 8% of the total hormone was labeled. If the voltage was allowed to reach 665 mV during the iodination process, 61 t 2%’ of the hormone was labeled, and this percentage did not increase with a further rise in voltage to 750 mV. Voltages higher than 800 mV result in oxidation and inactivation of the hormone (I). As might be expected, iodination efficiency was highly dependent upon the conTABLE VOI

I AGI- DI PE NDI

Maximum voltage (mV) 580 665 750

2

NC F- OF ‘2’I-L~~h~.~~~

EFFICIENCY

Percentage iodination i SEM

II

37.0 k 7.71 61.35 ? 1.66 56.6 f 5. II

z 2 3

PREPARATION

OF TABLE

CON~EN bPTH

IRA-I ION DEPENDENCE

3 OF ‘2JI-L,~~~~

Na”‘, NUWmoles

added (nmol)

40 20 IO

4.21 ?.I I I .05

4.21 2.11 I.05

bPTH

ING EFFICIYLNC.~ Concentration product [PTH][“‘I] ( x I or”‘)

added

Micrograms

71

[“‘I]IODO-bPTH

or “‘1 (hl)

2.81 x IO-” 1.40 x 10 i 7.02 x IO-”

” Values given are mean i SE of two determinations each. ’ Indicates significant difference (P < 0.05) when compared

centration of the reactants present. In exploring the effects of concentration, the molar ratio of iodine to bPTH was maintained at unity. With 40 c.Lgof bPTH present, the reaction proceeded until 59% of the hormone was radioactively tagged, whereas if this amount was decreased to 20 pg, the percentage of hormone present that was labeled fell to 47%. When 10 pg of bPTH was present the percentage of iodide incorporated fell to 23%. Reactant concentrations are thus a major consideration in selecting appropriate conditions for iodinating a given amount of hormone. Carrier-free [‘2”I]-labeled bPTH was prepared for bioas:say using the following conditions: pH 7.4, maximum voltage of 750 mV, bPTH, and Ya’““I at 4.5 nmol. In the final product, 60% of the iodide was associated with the intact bPTH. Prior to bioassay of the labeled hormone the free, unreacted iodide was separated from the reaction mixture by gel filtration on a column of Biogel P-2 (0.7 x I6 cm). The radioactive material eluting in the void volume was pooled and an aliquot subjected to gel filtration analysis. The results. shown in Fig. 2, confirmed that virtually all the radioactive material to be injected eluted at the position expected for intact bPTH; less than 2% eluted as free iodide. This preparation was assayed 24 h after iodination. Bioactivity of [““Iliodo-bPTH or an equal amount of unlabeled bPTH was assessed by its potency in elevating the serum calcium

7.90 I.96 0.493

with

Percentage iodination’l 59.2 i 4.1 47.9 i I.6 ‘3.9 - 3.9

50.2’;.

levels of chicks. Previous experiments (not shown) had established that normal serum Ca’+ levels were between IO and I1 mg%, and that on a log-dose response plot, the response was linear between doses of 2.5 U to 10 U per chick. The maximal response occurred at doses above this. These findings are in agreement with earlier reports (6,9). At doses of 2.5, 5, 8, and I I U per chick the labeled and unlabeled hormone preparations were equipotent. as established by a comparison of the slopes of the regression lines on a log-dose response plot (see Fig. 3). DISCUSSION The electrolytic method for iodinating proteins offers a rapid. easily controlled technique for preparing biologically active, radioiodine-labeled parathyroid hormone which is damage free (I 1. After modifying the apparatus to accommodate small amounts of hormone, the effects of variations in pH voltage and hormone concentrations on the efficiency of hormone iodination were investigated. Although pH variation might be expected to influence the iodine species present in solution (17). as well as the conformation of the polypeptide hormone itself, there were no detectable effects on the percentage of hormone iodinated over the range examined, pH 6.4 to 8.4. Lowering the voltage slowed the rate of generation of iodine and

72

NIELSEN

b 25

5 Log

Dose (Ulchlckl

8

I/

25

bPTH

FIG. 3. Log-dose response curve for it, V~\YI bioassay of [‘*“I]iodo-bYI’H (0) and unlabeled bPTH (0) at doses of I .O. 2.0. 3.2. and 4.4 g/chick. The uninjected controls, 10.8 t 0.1 is indicated by (W). Details are presented under Materials and Method\. The linear regressions (least squares method) for the two sets ofdata are indicated: (-1 controls, (- - -) iodinated product. There were no significant differences in slopes or intercepts. Also indicated, by the heavy line. is the linear regression to be expected if the hormone had been inactivated by radioiodination.

lowered the yield of hormone labeled. The voltage cannot be increased above 750 mV without incurring the risk of methionine oxidation and loss of bioactivity (I). Lowering the concentration of reactants markedly lowered the yield. In a bimolecular reaction of this type, the rate of reaction can be expected to be a function of the square of the hormone concentration. Apparently as the rate of hormone-iodine interaction is retarded, the relative importance of undesired side reactions (volatilization of I.,, conversion to IO:{-, etc.) increases. It is not feasible to increase the iodine concentration to compensate for this problem (15). Monoiodotyrosine is a more avid acceptor of iodine than is tyrosine itself (1). Therefore if the molar ratio of iodide:hormone were increased above unity, a higher proportion of the label would be in the form of diiodotyrosine (1). The possibility of iodination of histidine residues also arises.

t7

.-If.

To avoid these problems of ambiguity concerning the location of the label. then. conditions for the bioassay preparation were chosen to match those previously shown to produce monolabeled hormone (tyrosine at position 43) with only l_S(J of the iodine as diiodotyrosine (I ). Nonetheless. the reduction in reaction volume rem sulted in a slightly lowered yield, from 73 vs. 60%. With this lowered yield, it is likely that the proportion of label as diiodotyrosine is even less than 15%. At lower specific activities, the electrolytically labeled hormone has been shown to be fully active biologically in four different assay systems: CO, production by isolated mitochondria (1). resorption of rat tibia incubated irr t’itr-o (I), renal adenyl cyclase activation (19). and the Munson assay using intact, parathyroidectomized rats (1). The present high specific activity, carrier-free hormone preparation, 1300 Ciimmol. was approximately 60% of the theoretical maximum activity for a monoiodinated product. Nonetheless. there was no detectable loss of biological activity in the chick bioassay. Thus. the intense radioactivity of the carrier-free preparation does not of itself cause a detectable loss of activity when assayed shortly after iodination. Indeed. radiolysis of other hormones labeled to high specific activities has been reported (20.21). In these cases it has been shown that. when the radioactive iodine atom decayed. the hormone molecule to which it was bound was simultaneously destroyed. Because of this phenomenon, the biologic activity of the remaining molecule was not affected by the decay process nor did the specific activities of the preparation decrease with time, as might otherwise be expected. We have attempted to avoid any questions in this regard both by checking the biologic activity of the [‘?“I]iodo-bPTH i)l ~~ivo and also by using [““Iliodo-bPTH as soon as possible after iodination. It is clear that a few nanomoles of SPTH can be electrolytically radiolabeled with IZ31to a very high specific

PREPARATION

OF

activity without loss of biological activity it7 I~~\YJ. Since normal circulating levels of PTH are thought to be in the picomolar range (22-24). this technique should prove valuable in producing bioactive preparations suitable for it7 \xi~,tt or it7 I*ift’cj experiments done at high dilutions.

the generous Keutmann.

II.

Breurr.

12.

/VI/I. Acc,d. Niall. H. D.,

13.

IS.

gift

of

purilied

bPTH 16.

2.

3.

Sammon. Raisz. 1603.

P. J.. Brand. L. G. I 1973)

Rownblatt. Tregear.

251, 15% 164. Malbon. C. C. (1976)

Receptor Research pp. 533-563. Dekker. 4.

Sutcliffe. Pilcryk,

5.

Mosely.

W. F.. and 92, 1.596-

(Blecher. New

i/r Methods

M.. Yorb.

ed.)

J. S.. Martin. T. J.. Eisman. R. (19731 Bzrx.iic,iit. .I. 134, J. M..

B. W.. /J//or/,/~/r 6.

J. S.. Neuman. E/tcloc riuc~/ct~,y

M.. Goltzmann. D.. Keutman. H. T.. G. W.. and Potts. J. T.. Jr. (1976) ./.

HL~I. (‘h<,r,,. Zull. J. E. and

Martin.

and Tregear. (11. PlrYvirll.

(‘lip.

W..

.-l/rci/.

(19751

c’~/ci/:

Y. Duke. C. G.. and ,I<“?‘ 92, 463-470.

Kenny.

A. D. t lY7?)E~1cloc

Pott\. J. Q. M.. Act/d

T.. Jr.. Aurbach. and Sandoval. SC,;. C~S.4 54,

l743-

A.

7i.\.\rre

G. D.. (1965) 1751.

Erl’.

101-151. Wea\t. R. c‘.,

Ronan.

19.

93. DiBella,

Craig. 1.. C. 18, ‘69-293.

P. J.. Kc.\.

Catt.

? I.

Hi~~/dW.\. Scarpace.

K.

!iherwood. prcx. h’crt.

(lY70)

Sl\t

C. D. 71-716. J..

and /.

Ht,c.$,t?i

22,

of Chemistry

p. D-112.

and

T.

(lY741

Chum.

Yalow.

Rubber

R. S. I 1976)

J. V..

P..

Greene.

Miller.

E..

S. S.,

P~IJ~,.

,Yrrr.

A.

(1973)

Baukel.

-215. J. ( IY77)

and

AC-trcl.

.S(,.

/li(x./tir~i.

Elrchj~,~i,r(,/r~c\

1405. Mophadam. H.. Menren. G. D. (1961) it! The

E. D.. Parathyroid\

R.O.. and R.V. Talmage. ed\.). Charle\ C Thornab. Springfield.

23.

Parson\. J. A.. and tlA~,l‘/r~,i, 250. s-257.

24.

Parsons.

J. A..

Kafferty.

J. M.. Keutman. E. N.. and

Calcium-Regulating M. 0. Ouen\. 33-39.

i 1962)

Handbook

ed..

A<./,, 313. ?‘I P.. and Dcftos.

Cupp. H. D.. McPherwn.

Zanrlli. Callahan,

Hogan. D..

H. T.. N~all. H. D.. Reit, B.. Pawn\. Potts. J. T.. Jr. ( lY73) Eotlr~ ,.ir~olr~y\

l398-

(Creep. 203-13.

R..

G. D.. and Sherwood. f’rcq. I/cw,,r. Kc\.

13491353. F. P.. Dousa.

20.

18. vi/r,)/-

.Aurhxh. Kccc~~r

PK~u.

D. A.. and Mun\l)n. 3. 117% 1182.

B.. Strau\\. E.. and 25, 260-767. G. W.. Rietschoten.

Amaud. U.S.4 71.

22.

R. 11970)

67. 1862~1869. H. T.. Sauer.

F.. Aurbach. G. Jr. (1970) II~,/‘/“‘-S~,?,/I,~‘\ 351, l5861588.

ed.

PhyGc\. Cleveland.

Kcutman. J. A., and

C‘hcrrl.

Samrnon.

K.

IO.

and

B.

J. T.. Jr.. M. (1966)

Schneider. Diahete\ 18. Tregear.

and

C. J. ( lY7i)

F. N. ( 19681 M.

9.

Potty. Q.

101,

Pawn\. J. A.. Reit. B.. and Robinwn. O,~/~~(~~i,ri~l~~~~ 92, 454462.

and Lane. 241p250.

Jr..

.Sc,i. USA Keutman.

H.. and f/cJrr,/. Kcr.

17.

C. J.. Reit.

G. W. (19751 2. 549-557.

Borle. A. B., and Rigg:‘. 40, 339-344. 8. Neuman. W. F.. Neuman

Rxmus~en, I’ny.

and Co..

in

Vol.

J. A.. YI3-Yll.

T. J.. Rohinwn.

7.

B..

M. L.. Dawwn. Pott\. J. T.. /“IIJ~\;,J/. (‘/w/u.

REFERENCES I.

H.

14. Ta\h.jian. A. H., Jr.. Ontiye\. P. L. ( 1964) Hits /r(~~t/str~~

ACKNOWLEDGMENT We appreciate from Dr. Henry

73

[“‘I]IODO-bPTH

Evcerpta

Reit. B..

B.

( 1974)

Gray.

D.,

H. T.. Tregear. Potts. J. T.. Jr. Hormones

and J.A. Media.

pp. Ill %‘~r/l~w

Rat.

B..

G. W.. ( 1975) i/r

(Talmage. Parwn\, Amsterdam.

and

R. V.. ed\.).

pp.