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The relative stabilities of 67Ga complexes lactoferrin and transferrin at various pH's
The Relative Stabilities of 67Ga Complexes of Lactoferrin and Transferrin at Various pH’s R. E. WEINER,* G. J. SCHREIBER,P. B. HOFFERand T. SHANNON Yale University School of Medicine, Department of Diagnostic Radiology, 333 Cedar Street, New Haven, CT 06510, U.S.A. (Receityed26 Mtrrch 198 1) The ability of lactoferrin (Lf) to remove 67Ga originally bound to transferrin (Tf) and Tf to dissociate 67Ga originally bound to Lf was examined using- the equilibrium dialysis technique. When Tf-“Ga was challenged by Lf for 20 h, at pH 7.4, Lf dissociated a majority ( - 755:) of the “‘Ga bound to Tf. Increasing the incubation citrate ion concentration to 150pM did not significantly accelerate the dissociation of Tf-67Ga complex by Lf. This dissolution of the Tf-67Ga complex was a specific effect due competitive uptake of 67Ga by the Lf iron binding sites since neither human serum albumin nor iron-replete Lf caused any significant dissociation of 67Ga bound to Tf. In contrast, under similar conditions, Tf did not remove activity from preformed Lf-67Ga complex. Lowering the pH from 7.4 to 6.2 (to mimic conditions found in an abscess or tumor), had little influence on these results. The incubation pH did influence the absolute amount of the radioactivity bound to Tf and Lf. As the pH was lowered from 7.4 to 6.2, Lf-bound activity decreased slightly from 96 to 87”; while the fraction of Tf-bound radionuclide decreased from 77 to 63;;,, Thus, Lf present in a tumor or abscess site would be able to remove and retain the 67Ga activity brought to the site by Tf whether or not acidic conditions prevailed.
Introduction THE RADIONUCLIDE,67Ga is widely used as a diagnostic agent for tumor and abscess detection. Although the ixact mechanism of gallium localization is unknown, results from many laboratories suggest iron-binding molecules play an important role. Compared to other blood proteins, transferrin (Tf), the iron transport protein of et al, serum, has a high affinity for 67Ga (HARRIS and AISEN, 1975; VALLABHAJOSULA 1980). A number of investigators have shown that in a variety of in vitro tumor models, the presence of Tf stimulates the cellular uptake of (j7Ga (HARRIS and SEPHTON,1977; LARSON et al., 1979; NOUJAIM et al., 1979). WONG and associates (1980) have demonstrated similar findings in an in viuo model. Besides delivering 67Ga to the lesion, Tf may also be involved in sequestration at the site. The presence of Tf has been demonstrated in artificially induced abscesses (SHELDONet al., 1967) and in saliva from an individual with chronic parotitis (TABAK et al., 1978). Work from this laboratory and by others suggests that lacoferrin (Lf), another ironbinding glycoprotein somewhat similar to Tf, may participate in 67Ga accumulation. Lf has relatively high affinity for 67Ga (WEINER et al., 1979) although not as high as for iron. ‘j’Ga-Lf complex has been isolated from the tears (HOFFERet al., 1978) and breast milk (HOFFER et al., 1977) of patients injected with 67Ga, suggesting the formation of a 67Ga-Lf complex in vivo. Lf appears to be the major 67Ga binding protein in polymorphonuclear leukocytes (PMN’s) (WEINER et al., 1981a). PMN’s are known to localize in abscesses. TZEN and coworkers (1980) have shown in an animal model that i.m. injection of Lf caused localization of i.v. administered 67Ga citrate at the i.m. injection site. The presence of increased concentration of Lf has been demonstrated in a number of pathological lesions. For example, an elevation in Lf concentration has been found in a * For reprints contact R. E. Weiner, Ph.D.; Department of Diagnostic Radiology; The University of Kansas College of Health Sciences and Hospital; Rainbow Boulevard at 39th Street; Kansas City, Kansas 66103. N.M.B. B/4 --K
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variety of malignant lesions (LOISILLIERet al., 1971; RO~QUES et ul., 1970), tumor tissue known to be gallium positive on scan (HOFFERet ul., 1979). and a number of inflammatory processes (BENNETTand SKOSEY 1977; TABAK et ul., 1978; FIGARELLAer al., 1978). Considering Lf’s affinity for (j7Ga and its increased concentration in numerous galliumpositive lesions, it seems likely Lf is an important factor in the sequestration of 67Ga. It has been suggested that 67Ga transported to a tumor or abscess by Tf is bound at the site by Lf (HOFFER, 1980). Previous work suggests that Lf has higher affinity for (j7Ga than Tf (HOFFER et al., 1977). Near neutral pH, Lf bound about 77% of the activity compared to only 507; bound to Tf. This difference in “?A bound” activity became greater as the pH decreased. However, even though Lf appears to have a higher affinity for 67Ga it is possible that steric or other kinetic barriers might preclude a clinically relevan; transfer of 67Ga between the two macromolecules. For example, even though the stability constant measured for the iron-Tf complex is approximately lo6 times greater than for ferric EDTA, iron is transferred only slowly from EDTA to apo-Tf (BATESet al., 1967). Therefore, in the following studies we have examined the ability of Lf to (a) remove 67Ga from a preformed Tf-67Ga complex and (b) retain bound 67Ga when challenged by Tf.
Materials and methods Equilibrium dialysis (ED) experiments were performed using a dialyzer consisting of 5 teflon dialysis cells stacked together which rotates at constant speed. The unit was placed in a water bath to maintain the cells at 37°C. Each cell was divided into 2 chambers by a membrane sandwiched between the two halves of the dialysis cell. In each of the 5 cells, 1 ml of a TT or Lft solution in buffer was added to the right chamber while 1 ml of buffer containing 67Ga-citrate$ was placed in the left chamber. Care was taken to minimize the introduction of citrate from the commercial 67Ga-citrate preparation by using small volumes containing high activity. If necessary, an aliquot of a stock Nacitrate solution was added to achieve the desired citrate concentration. In a few experiments, the Tf-67Ga compex was formed and the solution dialyzed at 4’C against three changes of buffer before addition to the equilibrium dialyzer. This removed any6’Ga bound to low affinity sites on Tf and rid the preparation of citrate ions. The buffer used in all experiments was 50 mM hydroxyethyl-piperazine-N’-2-ethanesulfonic acid (HEPES)containing 1OOmM NaCl. After equilibrium was reached, 100~1 of a lo-times concentrated solution containing the competing protein, either Tf, Lf, human serum albumin (HSA) or holo-Lf (iron replete), was added to the left chamber and 100~1 of buffer was added to the right chamber of 3 cells. In the 2 other cells, 100 ~1 of buffer was added to both right and left chambers. At specified times, 20 ~1 was withdrawn from each chamber and the radioactivity measured. In the chambers with no added competing protein the fraction of the activity which was protein bound was calculated by standard methods (LUTZ ef al., 1973). However, in the competition experiments when protein was present in both chambers, the fraction of non-protein bound activity could not be directly ascertained. Therefore a relative amount of activity bound was computed and designated as “Y(ibound”. This parameter provided information about the shift of radionuclide from Tf to apo-Lf, holo-Lf or HSA and was defined by the equation: “~,~bound” = (R - L)/R -t L) x 100. Where R = activity in the right chamber which contained Tf and L = activity in the left chamber which initially contained only 67Ga and subsequently the competing protein. The “%bound” values from each cell were averaged. To determine the actual fraction of activity which was associated with the challenged Tf and the competing macromolecules after ED it was necessary to quantitate the “free” activity. After reaching equilibrium the samples were removed from the * Sigma Chemical Company. t Calbiochem-Beringer, Inc. $ New England Nuclear.
The relative .stuhili~ies of”G~
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and equal aliquots, 0.5-l ml, were chromatographed separately on 1.5 x 27 cm column containing G-50 Sephadex gel. Control experiments showed this procedure separated protein bound activity from the “free” activity or non-protein bound; greater than 900,:,of the added activity was recovered for each column. The amount of “free” and protein bound activity obtained from these columns was used to calculate the actual distribution of activity in each fraction. Experiments were routinely carried out for at least 24 h ; however, some were allowed to proceed 40 h. Since Lf and Tf have a high affinity for ferric and other metal ions, careful attention was paid to minimize spurious metal ion contamination in all equipment and reagents (WEINERet uI., 1979). Analysis of Tf and Lf solutions (HARRIS,1978) showed iron saturation for Tf and Lf to be 7.5”;, and 26”., respectively. The rate of dissociation (DR) was defined as fractional decrease in “%bound” activity per h and was calculated from the plots of “YAbound” VS equilibration time. The rate was equalled to the least squares slope of the line which included points between O-6 h after the addition of Lf (R > 0.95). To prepare the holo-Lf, and aliquot of freshly prepared Fe-nitrilotriacetic acid solution was added to a Lf solution to achieve a 20;; molar excess of Fe3’. After the sample was incubated overnight at 37°C non-bound iron was removed (WEINERet ul., 1979).
dialyzer
Results
Figure 1 shows the ED experimental results of the Tf-67Ga complex challenged by Lf experiments at two hydrogen ion concentrations. At pH 7.4, about 75:‘~:of the 67Ga activity bound to Tf in an hour (Fig. 1A). At the time indicated by the arrow, Lf was added to sample cells while only buffer was added to the control cells. Initially, the decrease in the ““;bound” in the cells where Lf was competing with Tf was rapid and large. That is, (j7Ga shifted from the right chamber which contained Tf to the left which contained Lf. This initial decrease probably represented Lf binding to free 67Ga ( . 25”,, before Lf addition). Any further decrease in “xbound” activity (below 50:/A)could only come from 67Ga dissociating from Tf. This occurred at about 1 l/2 h after the Lf addition. From 1 l/2 h to about 20 h, when equilibrium was reached, a real and large dissociation of the 67Ga from Tf and formation of the Lf-67Ga complex occurred. In contrast, the control ““/Lbound” activity value was relatively constant. To eliminate the possibility this dissociation represented transfer of 67Ga bound at low affinity sites on Tf, the Tf-67Ga complex was preformed before placement in the equilibrium dialyzer and then challenged by Lf in the presence of 15 PM citrate. Both the dissociation rate (- 17 f 39;) and the final “:,;bound”, (-67 + 47;) were comparable to the values obtained with the Tff6’Ga complex which was formed in the ED apparatus (see Fig. 1 and Table 1). These samples were also analyzed for actual “/i bound activity using chromatography on G-50 Sephadex gel. At equilibrium, a majority of activity (75 + 39,) was bound to Lf while only 16 + 4Y,; remained bound to Tf and 1.4 + 0.10, was “free” activity (not corrected for column losses). This contrasted sharply to the distribution of radioactivity in the unchallenged preformed Tf-67Ga where Tf bound 95.5 + 0.47: and 4.5 f 0.47; remained “free”. This suggests Lf removed a large fraction of the 67Ga bound to the high affinity Tf site. At pH, 7, 6.6 and 6.3 (Fig. lB), these qualitative results were the same. The only difference was that less 67Ga was bound to Tf at the lower incubation pH’s (cf. Figs 1A and 1B). Also, the pH did not appear to influence the rate of dissociation (Table 1A). A linear regression analysis of the data, pH VS rate of decrease, yielded a very low correlation coefficient, R = 0.204 which indicated no relationship between these factors. To insure this loss of 67Ga from Tf caused by the addition of Lf was a specific effect, two control experiments were performed. First, HSA at a comparable concentration, was
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FIG. 1. Equilibrium Dialysis of Tf-67Ga challenged by Lf at different pIi’s. At a time point indicated by the arrow, 100~1of Lf solution (A), or buffer (0) was added to the dialysis chambers. The points and error bars represent the mean and SEM, respectively. The line denoted by the stars represents a least squares plot of the data. The incubation temperature was 37°C and buffer was 50 mM HEPES containing 100mM NaCl. (A) Incubation pH = 7.4, conditions: 29 PM Tf, Lf 46 hM, 0.5 &i 67Ga per cell, and 27.2H citrate. (II). Incubation pH = 6.3, conditions: 25 pM Tf, 33.4PM Lf, 2.9 pCi ‘j’Ga per cell, and 13.6nM citrate.
added - .___.as the competing protein. There was no significant effect of the HSA addition on the amount of activity bound to Tf (data not shown). Second, a solution of Lf which had been saturated with iron was used to challenge the Tf. The addition of holo-Lf as a competitor caused little reduction in Tf-bound 67Ga (Fig. 2). There was a slight drop in the ““/,bound” activity value after 20 h. This may indicate the Lf was not lOOo/,saturated with iron and the unsaturated Lf was binding a small portion of the “free 67Ga” present. However, this slight diminution was not significant compared to the large rapid reduction which occurred with the relatively unsaturated Lf (cf. Fig. 1A and Fig. 2). Tf challenged by Lf; injiuence of citrate concentration Table 1B shows that increasing citrate concentration to 75 PM or 150 PM does not significantly increase the DR compared to the lower citrate concentration. Moreover, when the preformed Tf-67Ga was challenged by Lf in the abscence of citrate ions, there was no significant change in the DR (Table 1B). However, the transfer rate after 6 h slowed compared to the rate in the presence of citrate ions and sightly less aetivity was transferred to Lf (see above). The equilibrium distribution was, 31 +_ 2”/, and 62 + 4% bound to Tf and Lf respectively, with 1.9 + 0.1% “free”.
The relative stabilities of (j7Ga TABLE 1. The influence
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of pH and citrate concentration dissociation rate (DR) of Tf challenged by Lf
A. pH pH*t 7.4 7.0 6.6 6.3
on the
Dissociation rate$ (%h-‘) - 19.0 * 3.7 -21.1 -41.1 -25.7
B. Citrate
Citrate molarity*$ (PM) 0.011 17.0 75.0 150.0
Dissociation rate’j percent/hr - 13.7 -19.0 + 3.7(n = 6) - 30.8 f 2.8 (n = 2) -24.7 &-2.8(n = 2)
0.6 < p < 0.7 0.1
* Conditions: Tf/Lf = 0.67, l-5 &i 67Ga per cell, 50 mM HEPES buffer containing 100 mM NaCI, 37°C. t 10-27.2 pM citrate. $ Slope calculated from “%bound” activity VS equilibrium time plot using time points, O-6 h after Lf addition. $pH = 7.4. 11Preformed Tf-‘j’Ga complex with no added citrate, see Materials and Methods. Lf challenged by Tf influence of incubation pH
When the Lf-67Ga complex was allowed to form at pH 7.4 (Fig. 3A) and at pH 6.3 (Fig. 3B) the addition of Tf caused only a small reduction in the “%bound” activity compared to the control cells where only buffer was added. The small difference between the control and Tf containing cells probably represented “free67Ga” or non-Lf-bound 67Ga, bound to Tf. A reduction in the incubation pH did not change the ability of Lf to retain 67Ga when challenged by Tf (Fig. 3). However, unchallenged Lf bound slightly less 67Ga as the pH of the solution was reduced (cf. Fig. 3A and 3B).
Discussion These results confirm the previous inference (HOFFER et al., 1977) that Lf has a higher 67Ga avidity than Tf and demonstrates that, under the conditions studied, Lf can readily
Equtlibrium time,
h
FIG. 2. Equilibrium Dialysis of Tf-67Ga challenged by iron saturated Lf, At the time point
indicated by the arrow 100~1 of an iron saturated solution of Lf (A) or buffer (0) was added to the dialysis cells. Conditions: pH 7.4, 27.5 PM Tf, 25.4 /IM Lf, 1.5 @i 67Ga per cell, 17 PM citrate and all other conditions are as described in Fig. 1.
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FIG. 3. Equilibrium Dialysis of Lf-67Ga challenged by Tf at different pH’s. At time point indicated by the arrow 100~1 of a Tf solution (0) or buffer (A) was added to the dialysis cells. (A) Conditions: incubation pH = 7.4, 27.4 PM Lf, 25 PM Tf, 2.4 &i per cell and 5 PM citrate. (B) Conditions: incubation pH = 6.2, 27.5 PM Lf, 25.3 PM Tf, 1.3 &i 67Ga per cell, and 10.4 PM citrate all other conditions for A and B are as described in Fig. 1.
remove Tf-bound 67Ga. It is not likely that this transfer represented Lf binding of 67Ga removed from 67Ga bound to low affinity sites on Tf. Lf caused dissociation of gallium from Tf whether the Tf-67Ga complex was preformed or formed in the ED apparatus. Therefore the exchange probably represented a translocation of 67Ga between the iron binding sites of both proteins. Experimental evidence has suggested gallium binds at the Tf-iron binding site with high affinity (HARRISand AISEN, 1975; RAISZADEW et al., 1981; KULPRATHIPANJA ef al., 1979). Work from this laboratory supports the concept that with Lf, gallium binds to the iron binding site (WEINERet al., 1978). Moreover, the inability of holo-Lf to remove Tf bound 67Ga is consistent with an exchange between iron binding sites. The ability of Lf to dissociate Tf bound 67Ga was not unexpected since Lf has 300 times greater affinity for iron than Tf (AISENand LIEBMAN,1972) and apo-Lf can rapidly remove iron from Tf under the appropriate conditions (VAN SNICK et al., 1974). The higher affinity of 67Ga for Lf compared to Tf implies the formation constant for the Lf-Ga complex is at least 1022.3, the value obtained for the Tf-Ga complex (KULPRATHIPANJAet a/., 1979). More importantly in terms of Lf’s role in gallium localization was the speed of the exchange between the two macromolecules. Metal ion exchange even though thermodynamically favored, may not be kinetically labile (BATESet al., 1967). However, the Tf-Ga complex was sufficiently labile to allow transfer to Lf within a clinically relevant time frame (hours rather than days). This type of rapid exchange of gallium with a chelate has also been observed with siderophores, the bacterial and fungal iron transport molecules (EMERYand HOFFER,1980). Gallium was able to rapidly replace (C~,~about 1 h at pH 6.0) some of the iron complexed to different siderophores even though the formation constants for these complexes are about 103o. The labile character of the gallium-Tf complex may be related to the fast exchange of the bicarbonate (carbonate) ion since this ion is thought to be directly coordinated to the metal ion (HARRISet al.,, 1974). Thus readily exchangable bicarbonate would be interconnected with an exchangable gallium atom. In contrast, the bicarbonate of the stable Fe-Tf complex does nor have a significant rate of exchange of bound with free bicarbonate in solution.
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The data presented in this paper are consistent with the findings of TZEN et al. (1980). The localization of 67Ga was studied with an i.v. injection of either preformed Tf-67Ga or 6’Ga-citrate in a rabbit. Histamine and Tf injected i.m. into the rabbit’s thigh did not cause any localization of the radionuclide while an i.m. injection of Lf caused a slight accumulation with the preformed Tf-67Ga complex. Accumulation of 67Ga by Lf or Tf after an i.v. injection of 67Ga was directly related to the iron binding capacity of these proteins. Increasing the iron saturation of Lf or Tf in a stepwise manner caused a concommitant reduction in the 67Ga accumulation. Therefore, at least in their animal model, Lf removed and retained Tf-bound 67Ga circulating in the blood and these results further support the concept that ‘j’Ga is bound to the iron binding site on Lf. Since the pH of abscess exudate (MURPHY, 1976; VAN SNICK el al., 1974) and the intracellular pH of certain tumors (POOLE, 1967; KAHLER and ROBERTSON,1943) is thought to be mildly acidic, the ability of Lf to dissociate ‘j7Ga bound Tf was studied at a variety of pH’s. Our results indicate that pH was not a controlling factor in the translocation of the radionuclide under the conditions studied. This result is surprising since Tf is known to have a lower affinity for 67Ga in the acid pH range (HOFFER ef al., 1977; RAISZADEHet al., 1980). However, though the off rate for the Ga-Tf complex was probably increased at lower pH the rate-limiting factor in these experiments may have been (j7Ga diffusion through the dialysis membrane. It has been observed that the kinetics of iron transfer from ferritin to Tf are slowed in the presence of a dialysis membrane (HARRIS, 1978). The addition of citrate and other small chelate ions to an incubation medium is known to dramatically increase the rate of transfer of iron from Tf to other molecules such as Lf (VAN SNICK et al., 1974) or desferrioxamine (POLLACK et al., 1976; MORGAN, 1979). Therefore it was also unexpected that increased concentration of citrate ions, at least up to 150 PM, appeared to have no influence on the rate of transfer. Moreover the initial transfer rate was unaffected in the abscence of citrate. This suggests gallium hydrolysis species were important in radionuclide translocation. The probable function of citrate would be to form additional soluble species at these concentrations while this ion may increase transfer only at higher concentrations (1 mM). Thus, citrate would probably not be important in in uivo localization because the concentration in tumors and abscesses would likely be comparable to blood concentration, 75 PM (WOLCOTTand BOYER,1948). A more important factor in mediating transfer of ‘j’Ga between macromolecules may be phosphate-containing compounds. Most recently we (WEINERet al., 1981b) have shown that ATP was the most effective compound mediating transfer of 67Ga from Tf to ferritin. Acknowledgements-the excellent typing of R. A. Cherlin is gratefully acknowledged. This research was supported by DOE Contract No. DE-AC02-78EVO4625.
References AISENP. and LEIBMAN A. Biochim. Biophys. Acta 257, 314 (1972). BATESG. W., BILLUPSC. and SALTMANP. J. Biol. Chem. 242, 2816 (1967). BENNETT. R. M. and SKOSEYJ. L. Arthritis Rheum. 20, 84 (1977). EMERYT. and HOFFERP. B. J. nucl. Med. 21, 935 (1980). FIGARELLA C., ESTEVENXIN J. P. and SARLESH. Lancer 1, 1105 (1978). HARRISA. W. and SEPHTON R. G. Cancer Res. 37, 3634 (1977). HARRISD. C. Biochemistry 17, 3071 (1978). HARRISD. C. and AISENP. In Proteins of Iron Storage and Transport in Biochemistry and Medicine (Edited by CRIGHTONR. R.) pp. 59-66. North-Holland Publishing Co., Amsterdam (1975). HARRISD. C., GRAY G. A. and AISENP. A. J. Biol. Chem. 249, 5261 (1974). HOFFERP. B. J. nucl. Med. 21, 282 (1980). HOFFERP. B., MILLER-CATCHPOLE R. and TURNERD. A. J. nucl. Med. 20, 424 (1979). HOFFERP. B., HUBERTYJ. and SAMUELA. M. J. nucl. Med. 19, 732 (abst) (1978). HOFFERP. B., HUBERTYJ. and KHAYAM-BASHI H. J. nucl. Med. 18, 713 (1977). KAHLERH. and ROBERTSON W. B. J. natn. Cancer Inst. 3,495 (1943). KLJLPRATHIPANJA S.. HNATOWICH D. J., BEHR. and ELMALEH D. Znt. J. nucl. Med. Biol. 6, 138 (1979).
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LARSONS. M., RASEYJ. S., ALLEND. R., NELXINN. J., GRUNBAUMZ., HARP G. D. and WILLIAMS D. L. .I. mtn. Cancer Inst. 64, 41 (1980). L~ISILIERF., POZZUOLIR. and BURTINP. Patho.-Viol. 19, 167 (1971). LUTZ R. A., LUTZ-EWANL. and WEDERH. G. Steroids 21,423 (1973). MORGANE. H. Biochim. Biophys. Acta 580, 312 (1979). MURPHYP. The Neutrophil p. 88. Plenum, New York (1976). NOUJAIMA. A., LENTLEB. C., HILL J. R., TERNERU. K. and WONG H. Int. J. nucl. Med. Viol. 6, 193 (1979). POLLOCKS., AISENP., LASKYF. D. and VANDERHOFF G. Br. J. Haemutol. 34,231 (1976). POOLED. T. J. Biol. Chem. 242, 3731 (1967). RAI~ZADEH M., HARWIGJ. F. and WOLF W. J. Lab. Comp. Radiopharm. 18, 167 (abst) (1981). ROUQLJES R., LOISILLIER F. and BURTINP. Rev. Franc. Etudes Clin. Biol. 15, 299 (1970). SHELDONW. H., MILDVAND. and ALLENJ. C. Jdhns Hopkins Med. J. 121, 113 (1967). TABAKL., MANDELI. D., HERRERAM. and BAURMASH J. Oral. Path. 7,91 (1978). TYMPERT. and GUTMANNW. 2. Gastroenterol. 17, 858 (1979). TZEN K-Y., OSTERZ. H., WAGNERH. N., JR. and TSAN M. F. J. nucl. Med. 21, 31 (1980). V~LLABHAJOSULA S. R., HARWIGJ. F., SIEMSEN J. K. and WOLF W. J. nucl. Med. 21,650 (1980). VAN SNICKJ. L., MA%~N P. L. and HEREMANS J. F. J. Exp. Med. 140, 1068 (1974). WEINERR. E., HOUR P. B. and THAKURM. L. J. nucl. Med. 22, 32 (1981a). WEINERR. E., SCHREIBER G. J. and HOFFERP. B. J. nucl. Med. 22, P33 (1981). WEINERR. E., THAKURM. L., GOODMANM. and HOFFERP. B. hoc. 2nd International Symposium on Radiopharmaceuticals pp. 331-340 Society of Nuclear Medicine, New York (1979). WEINERR. E., HOFFEXP. B. and THAKUR M. L. Proc. Second International Congress of the Worfd Federation of Nuclear Medicine and Biology p. 73 (abst). September 1978, Washington, D.C. (1978). WOLCOTTG. H. and B~VERP. D. J. Biol. Gem. 172,729 (1948). WONG H., TERNERU. K., ENGLISHD., NOUJAIMA. A., LENTLEB. C. and HILL J. R. Int. J. nucl. Med. Biol. 7, 9 (1980).
Introduction THE RADIONUCLIDE,67Ga is widely used as a diagnostic agent for tumor and abscess detection. Although the ixact mechanism of gallium localization is unknown, results from many laboratories suggest iron-binding molecules play an important role. Compared to other blood proteins, transferrin (Tf), the iron transport protein of et al, serum, has a high affinity for 67Ga (HARRIS and AISEN, 1975; VALLABHAJOSULA 1980). A number of investigators have shown that in a variety of in vitro tumor models, the presence of Tf stimulates the cellular uptake of (j7Ga (HARRIS and SEPHTON,1977; LARSON et al., 1979; NOUJAIM et al., 1979). WONG and associates (1980) have demonstrated similar findings in an in viuo model. Besides delivering 67Ga to the lesion, Tf may also be involved in sequestration at the site. The presence of Tf has been demonstrated in artificially induced abscesses (SHELDONet al., 1967) and in saliva from an individual with chronic parotitis (TABAK et al., 1978). Work from this laboratory and by others suggests that lacoferrin (Lf), another ironbinding glycoprotein somewhat similar to Tf, may participate in 67Ga accumulation. Lf has relatively high affinity for 67Ga (WEINER et al., 1979) although not as high as for iron. ‘j’Ga-Lf complex has been isolated from the tears (HOFFERet al., 1978) and breast milk (HOFFER et al., 1977) of patients injected with 67Ga, suggesting the formation of a 67Ga-Lf complex in vivo. Lf appears to be the major 67Ga binding protein in polymorphonuclear leukocytes (PMN’s) (WEINER et al., 1981a). PMN’s are known to localize in abscesses. TZEN and coworkers (1980) have shown in an animal model that i.m. injection of Lf caused localization of i.v. administered 67Ga citrate at the i.m. injection site. The presence of increased concentration of Lf has been demonstrated in a number of pathological lesions. For example, an elevation in Lf concentration has been found in a * For reprints contact R. E. Weiner, Ph.D.; Department of Diagnostic Radiology; The University of Kansas College of Health Sciences and Hospital; Rainbow Boulevard at 39th Street; Kansas City, Kansas 66103. N.M.B. B/4 --K
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variety of malignant lesions (LOISILLIERet al., 1971; RO~QUES et ul., 1970), tumor tissue known to be gallium positive on scan (HOFFERet ul., 1979). and a number of inflammatory processes (BENNETTand SKOSEY 1977; TABAK et ul., 1978; FIGARELLAer al., 1978). Considering Lf’s affinity for (j7Ga and its increased concentration in numerous galliumpositive lesions, it seems likely Lf is an important factor in the sequestration of 67Ga. It has been suggested that 67Ga transported to a tumor or abscess by Tf is bound at the site by Lf (HOFFER, 1980). Previous work suggests that Lf has higher affinity for (j7Ga than Tf (HOFFER et al., 1977). Near neutral pH, Lf bound about 77% of the activity compared to only 507; bound to Tf. This difference in “?A bound” activity became greater as the pH decreased. However, even though Lf appears to have a higher affinity for 67Ga it is possible that steric or other kinetic barriers might preclude a clinically relevan; transfer of 67Ga between the two macromolecules. For example, even though the stability constant measured for the iron-Tf complex is approximately lo6 times greater than for ferric EDTA, iron is transferred only slowly from EDTA to apo-Tf (BATESet al., 1967). Therefore, in the following studies we have examined the ability of Lf to (a) remove 67Ga from a preformed Tf-67Ga complex and (b) retain bound 67Ga when challenged by Tf.
Materials and methods Equilibrium dialysis (ED) experiments were performed using a dialyzer consisting of 5 teflon dialysis cells stacked together which rotates at constant speed. The unit was placed in a water bath to maintain the cells at 37°C. Each cell was divided into 2 chambers by a membrane sandwiched between the two halves of the dialysis cell. In each of the 5 cells, 1 ml of a TT or Lft solution in buffer was added to the right chamber while 1 ml of buffer containing 67Ga-citrate$ was placed in the left chamber. Care was taken to minimize the introduction of citrate from the commercial 67Ga-citrate preparation by using small volumes containing high activity. If necessary, an aliquot of a stock Nacitrate solution was added to achieve the desired citrate concentration. In a few experiments, the Tf-67Ga compex was formed and the solution dialyzed at 4’C against three changes of buffer before addition to the equilibrium dialyzer. This removed any6’Ga bound to low affinity sites on Tf and rid the preparation of citrate ions. The buffer used in all experiments was 50 mM hydroxyethyl-piperazine-N’-2-ethanesulfonic acid (HEPES)containing 1OOmM NaCl. After equilibrium was reached, 100~1 of a lo-times concentrated solution containing the competing protein, either Tf, Lf, human serum albumin (HSA) or holo-Lf (iron replete), was added to the left chamber and 100~1 of buffer was added to the right chamber of 3 cells. In the 2 other cells, 100 ~1 of buffer was added to both right and left chambers. At specified times, 20 ~1 was withdrawn from each chamber and the radioactivity measured. In the chambers with no added competing protein the fraction of the activity which was protein bound was calculated by standard methods (LUTZ ef al., 1973). However, in the competition experiments when protein was present in both chambers, the fraction of non-protein bound activity could not be directly ascertained. Therefore a relative amount of activity bound was computed and designated as “Y(ibound”. This parameter provided information about the shift of radionuclide from Tf to apo-Lf, holo-Lf or HSA and was defined by the equation: “~,~bound” = (R - L)/R -t L) x 100. Where R = activity in the right chamber which contained Tf and L = activity in the left chamber which initially contained only 67Ga and subsequently the competing protein. The “%bound” values from each cell were averaged. To determine the actual fraction of activity which was associated with the challenged Tf and the competing macromolecules after ED it was necessary to quantitate the “free” activity. After reaching equilibrium the samples were removed from the * Sigma Chemical Company. t Calbiochem-Beringer, Inc. $ New England Nuclear.
The relative .stuhili~ies of”G~
313
and equal aliquots, 0.5-l ml, were chromatographed separately on 1.5 x 27 cm column containing G-50 Sephadex gel. Control experiments showed this procedure separated protein bound activity from the “free” activity or non-protein bound; greater than 900,:,of the added activity was recovered for each column. The amount of “free” and protein bound activity obtained from these columns was used to calculate the actual distribution of activity in each fraction. Experiments were routinely carried out for at least 24 h ; however, some were allowed to proceed 40 h. Since Lf and Tf have a high affinity for ferric and other metal ions, careful attention was paid to minimize spurious metal ion contamination in all equipment and reagents (WEINERet uI., 1979). Analysis of Tf and Lf solutions (HARRIS,1978) showed iron saturation for Tf and Lf to be 7.5”;, and 26”., respectively. The rate of dissociation (DR) was defined as fractional decrease in “%bound” activity per h and was calculated from the plots of “YAbound” VS equilibration time. The rate was equalled to the least squares slope of the line which included points between O-6 h after the addition of Lf (R > 0.95). To prepare the holo-Lf, and aliquot of freshly prepared Fe-nitrilotriacetic acid solution was added to a Lf solution to achieve a 20;; molar excess of Fe3’. After the sample was incubated overnight at 37°C non-bound iron was removed (WEINERet ul., 1979).
dialyzer
Results
Figure 1 shows the ED experimental results of the Tf-67Ga complex challenged by Lf experiments at two hydrogen ion concentrations. At pH 7.4, about 75:‘~:of the 67Ga activity bound to Tf in an hour (Fig. 1A). At the time indicated by the arrow, Lf was added to sample cells while only buffer was added to the control cells. Initially, the decrease in the ““;bound” in the cells where Lf was competing with Tf was rapid and large. That is, (j7Ga shifted from the right chamber which contained Tf to the left which contained Lf. This initial decrease probably represented Lf binding to free 67Ga ( . 25”,, before Lf addition). Any further decrease in “xbound” activity (below 50:/A)could only come from 67Ga dissociating from Tf. This occurred at about 1 l/2 h after the Lf addition. From 1 l/2 h to about 20 h, when equilibrium was reached, a real and large dissociation of the 67Ga from Tf and formation of the Lf-67Ga complex occurred. In contrast, the control ““/Lbound” activity value was relatively constant. To eliminate the possibility this dissociation represented transfer of 67Ga bound at low affinity sites on Tf, the Tf-67Ga complex was preformed before placement in the equilibrium dialyzer and then challenged by Lf in the presence of 15 PM citrate. Both the dissociation rate (- 17 f 39;) and the final “:,;bound”, (-67 + 47;) were comparable to the values obtained with the Tff6’Ga complex which was formed in the ED apparatus (see Fig. 1 and Table 1). These samples were also analyzed for actual “/i bound activity using chromatography on G-50 Sephadex gel. At equilibrium, a majority of activity (75 + 39,) was bound to Lf while only 16 + 4Y,; remained bound to Tf and 1.4 + 0.10, was “free” activity (not corrected for column losses). This contrasted sharply to the distribution of radioactivity in the unchallenged preformed Tf-67Ga where Tf bound 95.5 + 0.47: and 4.5 f 0.47; remained “free”. This suggests Lf removed a large fraction of the 67Ga bound to the high affinity Tf site. At pH, 7, 6.6 and 6.3 (Fig. lB), these qualitative results were the same. The only difference was that less 67Ga was bound to Tf at the lower incubation pH’s (cf. Figs 1A and 1B). Also, the pH did not appear to influence the rate of dissociation (Table 1A). A linear regression analysis of the data, pH VS rate of decrease, yielded a very low correlation coefficient, R = 0.204 which indicated no relationship between these factors. To insure this loss of 67Ga from Tf caused by the addition of Lf was a specific effect, two control experiments were performed. First, HSA at a comparable concentration, was
R. E. Weiner et al.
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60
= 0
40
#j
20
8
0
=
-20 -40
Equilibrium T
I
1
I
time,
I
h
I,,
I
I
B 60
60
40 =
P
20
a a
O
s =
-20 -40 -60
L Equilibrlum
time
,h
FIG. 1. Equilibrium Dialysis of Tf-67Ga challenged by Lf at different pIi’s. At a time point indicated by the arrow, 100~1of Lf solution (A), or buffer (0) was added to the dialysis chambers. The points and error bars represent the mean and SEM, respectively. The line denoted by the stars represents a least squares plot of the data. The incubation temperature was 37°C and buffer was 50 mM HEPES containing 100mM NaCl. (A) Incubation pH = 7.4, conditions: 29 PM Tf, Lf 46 hM, 0.5 &i 67Ga per cell, and 27.2H citrate. (II). Incubation pH = 6.3, conditions: 25 pM Tf, 33.4PM Lf, 2.9 pCi ‘j’Ga per cell, and 13.6nM citrate.
added - .___.as the competing protein. There was no significant effect of the HSA addition on the amount of activity bound to Tf (data not shown). Second, a solution of Lf which had been saturated with iron was used to challenge the Tf. The addition of holo-Lf as a competitor caused little reduction in Tf-bound 67Ga (Fig. 2). There was a slight drop in the ““/,bound” activity value after 20 h. This may indicate the Lf was not lOOo/,saturated with iron and the unsaturated Lf was binding a small portion of the “free 67Ga” present. However, this slight diminution was not significant compared to the large rapid reduction which occurred with the relatively unsaturated Lf (cf. Fig. 1A and Fig. 2). Tf challenged by Lf; injiuence of citrate concentration Table 1B shows that increasing citrate concentration to 75 PM or 150 PM does not significantly increase the DR compared to the lower citrate concentration. Moreover, when the preformed Tf-67Ga was challenged by Lf in the abscence of citrate ions, there was no significant change in the DR (Table 1B). However, the transfer rate after 6 h slowed compared to the rate in the presence of citrate ions and sightly less aetivity was transferred to Lf (see above). The equilibrium distribution was, 31 +_ 2”/, and 62 + 4% bound to Tf and Lf respectively, with 1.9 + 0.1% “free”.
The relative stabilities of (j7Ga TABLE 1. The influence
375
of pH and citrate concentration dissociation rate (DR) of Tf challenged by Lf
A. pH pH*t 7.4 7.0 6.6 6.3
on the
Dissociation rate$ (%h-‘) - 19.0 * 3.7 -21.1 -41.1 -25.7
B. Citrate
Citrate molarity*$ (PM) 0.011 17.0 75.0 150.0
Dissociation rate’j percent/hr - 13.7 -19.0 + 3.7(n = 6) - 30.8 f 2.8 (n = 2) -24.7 &-2.8(n = 2)
0.6 < p < 0.7 0.1
* Conditions: Tf/Lf = 0.67, l-5 &i 67Ga per cell, 50 mM HEPES buffer containing 100 mM NaCI, 37°C. t 10-27.2 pM citrate. $ Slope calculated from “%bound” activity VS equilibrium time plot using time points, O-6 h after Lf addition. $pH = 7.4. 11Preformed Tf-‘j’Ga complex with no added citrate, see Materials and Methods. Lf challenged by Tf influence of incubation pH
When the Lf-67Ga complex was allowed to form at pH 7.4 (Fig. 3A) and at pH 6.3 (Fig. 3B) the addition of Tf caused only a small reduction in the “%bound” activity compared to the control cells where only buffer was added. The small difference between the control and Tf containing cells probably represented “free67Ga” or non-Lf-bound 67Ga, bound to Tf. A reduction in the incubation pH did not change the ability of Lf to retain 67Ga when challenged by Tf (Fig. 3). However, unchallenged Lf bound slightly less 67Ga as the pH of the solution was reduced (cf. Fig. 3A and 3B).
Discussion These results confirm the previous inference (HOFFER et al., 1977) that Lf has a higher 67Ga avidity than Tf and demonstrates that, under the conditions studied, Lf can readily
Equtlibrium time,
h
FIG. 2. Equilibrium Dialysis of Tf-67Ga challenged by iron saturated Lf, At the time point
indicated by the arrow 100~1 of an iron saturated solution of Lf (A) or buffer (0) was added to the dialysis cells. Conditions: pH 7.4, 27.5 PM Tf, 25.4 /IM Lf, 1.5 @i 67Ga per cell, 17 PM citrate and all other conditions are as described in Fig. 1.
376
R. E. Weiner
Equilibrium
time,
et al.
h
FIG. 3. Equilibrium Dialysis of Lf-67Ga challenged by Tf at different pH’s. At time point indicated by the arrow 100~1 of a Tf solution (0) or buffer (A) was added to the dialysis cells. (A) Conditions: incubation pH = 7.4, 27.4 PM Lf, 25 PM Tf, 2.4 &i per cell and 5 PM citrate. (B) Conditions: incubation pH = 6.2, 27.5 PM Lf, 25.3 PM Tf, 1.3 &i 67Ga per cell, and 10.4 PM citrate all other conditions for A and B are as described in Fig. 1.
remove Tf-bound 67Ga. It is not likely that this transfer represented Lf binding of 67Ga removed from 67Ga bound to low affinity sites on Tf. Lf caused dissociation of gallium from Tf whether the Tf-67Ga complex was preformed or formed in the ED apparatus. Therefore the exchange probably represented a translocation of 67Ga between the iron binding sites of both proteins. Experimental evidence has suggested gallium binds at the Tf-iron binding site with high affinity (HARRISand AISEN, 1975; RAISZADEW et al., 1981; KULPRATHIPANJA ef al., 1979). Work from this laboratory supports the concept that with Lf, gallium binds to the iron binding site (WEINERet al., 1978). Moreover, the inability of holo-Lf to remove Tf bound 67Ga is consistent with an exchange between iron binding sites. The ability of Lf to dissociate Tf bound 67Ga was not unexpected since Lf has 300 times greater affinity for iron than Tf (AISENand LIEBMAN,1972) and apo-Lf can rapidly remove iron from Tf under the appropriate conditions (VAN SNICK et al., 1974). The higher affinity of 67Ga for Lf compared to Tf implies the formation constant for the Lf-Ga complex is at least 1022.3, the value obtained for the Tf-Ga complex (KULPRATHIPANJAet a/., 1979). More importantly in terms of Lf’s role in gallium localization was the speed of the exchange between the two macromolecules. Metal ion exchange even though thermodynamically favored, may not be kinetically labile (BATESet al., 1967). However, the Tf-Ga complex was sufficiently labile to allow transfer to Lf within a clinically relevant time frame (hours rather than days). This type of rapid exchange of gallium with a chelate has also been observed with siderophores, the bacterial and fungal iron transport molecules (EMERYand HOFFER,1980). Gallium was able to rapidly replace (C~,~about 1 h at pH 6.0) some of the iron complexed to different siderophores even though the formation constants for these complexes are about 103o. The labile character of the gallium-Tf complex may be related to the fast exchange of the bicarbonate (carbonate) ion since this ion is thought to be directly coordinated to the metal ion (HARRISet al.,, 1974). Thus readily exchangable bicarbonate would be interconnected with an exchangable gallium atom. In contrast, the bicarbonate of the stable Fe-Tf complex does nor have a significant rate of exchange of bound with free bicarbonate in solution.
The relatice
stuhilities
of 6’G~
311
The data presented in this paper are consistent with the findings of TZEN et al. (1980). The localization of 67Ga was studied with an i.v. injection of either preformed Tf-67Ga or 6’Ga-citrate in a rabbit. Histamine and Tf injected i.m. into the rabbit’s thigh did not cause any localization of the radionuclide while an i.m. injection of Lf caused a slight accumulation with the preformed Tf-67Ga complex. Accumulation of 67Ga by Lf or Tf after an i.v. injection of 67Ga was directly related to the iron binding capacity of these proteins. Increasing the iron saturation of Lf or Tf in a stepwise manner caused a concommitant reduction in the 67Ga accumulation. Therefore, at least in their animal model, Lf removed and retained Tf-bound 67Ga circulating in the blood and these results further support the concept that ‘j’Ga is bound to the iron binding site on Lf. Since the pH of abscess exudate (MURPHY, 1976; VAN SNICK el al., 1974) and the intracellular pH of certain tumors (POOLE, 1967; KAHLER and ROBERTSON,1943) is thought to be mildly acidic, the ability of Lf to dissociate ‘j7Ga bound Tf was studied at a variety of pH’s. Our results indicate that pH was not a controlling factor in the translocation of the radionuclide under the conditions studied. This result is surprising since Tf is known to have a lower affinity for 67Ga in the acid pH range (HOFFER ef al., 1977; RAISZADEHet al., 1980). However, though the off rate for the Ga-Tf complex was probably increased at lower pH the rate-limiting factor in these experiments may have been (j7Ga diffusion through the dialysis membrane. It has been observed that the kinetics of iron transfer from ferritin to Tf are slowed in the presence of a dialysis membrane (HARRIS, 1978). The addition of citrate and other small chelate ions to an incubation medium is known to dramatically increase the rate of transfer of iron from Tf to other molecules such as Lf (VAN SNICK et al., 1974) or desferrioxamine (POLLACK et al., 1976; MORGAN, 1979). Therefore it was also unexpected that increased concentration of citrate ions, at least up to 150 PM, appeared to have no influence on the rate of transfer. Moreover the initial transfer rate was unaffected in the abscence of citrate. This suggests gallium hydrolysis species were important in radionuclide translocation. The probable function of citrate would be to form additional soluble species at these concentrations while this ion may increase transfer only at higher concentrations (1 mM). Thus, citrate would probably not be important in in uivo localization because the concentration in tumors and abscesses would likely be comparable to blood concentration, 75 PM (WOLCOTTand BOYER,1948). A more important factor in mediating transfer of ‘j’Ga between macromolecules may be phosphate-containing compounds. Most recently we (WEINERet al., 1981b) have shown that ATP was the most effective compound mediating transfer of 67Ga from Tf to ferritin. Acknowledgements-the excellent typing of R. A. Cherlin is gratefully acknowledged. This research was supported by DOE Contract No. DE-AC02-78EVO4625.
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