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Sephadex filtration of serum iodocompounds and protein-binding of iodotyrosines
CLINICA
CCA
CHIMICA
285
ACTA
4512
SEPHADEX BINDING
JANINE
FILTRATION
BISMUTH,
Laboratoive de Biochimie Sante’ et de la Rechevche rpMavseille 5 (France) (Received
OF SERUM
IODOCOMPOUNDS
AND
PROTEIN-
OF IODOTYROSINES”
April
MATHILDE Mkdicale Mgdicale,
CASTAY
AND SERGE
et Unite’ thyroi’dienne Faculte’ de Me’decine.
LISSITZKY
de l’lnstitut National 2 7 Bd. Jealz-Moulin,
de la
8, 1971)
SUMMARY
Di-iodotyrosine binding to serum protein cannot be demonstrated either by electrophoresis on paper, cellulose acetate or acrylamide-agarose (using various buffers at different pHs) or by double diffusion in agar and immunoelectrophoresis. To the contrary, binding of DIT to serum protein can be estimated by Sephadex G-25 gel filtration and by adsorption on charcoal. Binding increases with pH between 6 and IO. It is almost negligible below pH 6. This property was used to propose a method of separation of all the known iodocompounds found in serum using a single serum sample and two consecutive filtrations on Sephadex G-25. The first filtration at pH 5.80 allows the separation of MIT from DIT and iodide and from PBI. The latter is resolved into T4, T, and iodoprotein by refiltration in 0.01 N NaOH. Recoveries of the radioactive iodine-labeled iodocompounds are almost quantitative. Sephadex gel filtration is useful in estimating the iodotyrosine content of serum. Experiments on DIT clearance from the blood in man support the idea that iodotyrosines are not normal components of the blood of euthyroid humans. The usefulness of Sephadex G-25 gel filtration in 0.01 N NaOH for the quantitative estimation of serum total hormonal iodine is confirmed.
Gel filtration on Sephadex at a pH near neutrality has been used to separate iodide from iodothyronines bound to serum proteins as well as from free iodothyronines1-3. Further studies showed that when equilibrated in diluted NaOH the method allowed to separate T4 and T, from iodide4-5. In these experimental conditions free iodotyrosines are eluted along with iodide whereas iodinated proteins, when present, are recovered in the void volume of the column. Sephadex filtration at pH 5.80 (ref. 6) separated DIT from MIT and iodide but left unresolved the iodine-containing material * This study was supported by the Commissariat & 1’Energie Atomique the Centre National de la Recherche Scientifique (Paris, France). Abbreviations: MIT, 3.iodotyrosine; DIT, thyroxine; SDS, sodium dodecylsulphate.
x,5-di-iodotyrosine;
T,,
(Saclay,
France)
x,5,3’-tri-iodothyronine;
Clin. Chin<. Acta,
35 (1971)
and by T,,
285-298
286
BISMUTH et cd.
excluded from the gel which contains protein-bound iodothyronines proteins. Serum protein binding of iodotyrosines, if any, can interfere
and iodinated with the inter-
pretation of the results of gel filtration methods used to resolve serum iodinated compounds. No systematic and conclusive investigation has been performed on the distribution of free and protein-bound serum iodotyrosines (see ref. 7 for a review). Study of the distribution of iodo-compounds in the blood is of fundamental importance for the diagnosis of thyroid diseases in man. The need of a simple, accurate and reproducible method allowing the separation of all the known serum iodocompounds is therefore obvious. In the present communication we describe a method of separation of the presently known iodocompounds by filtration on Sephadex G-25 equilibrated at two different pHs and using a single serum sample. The validity of the method has been ascertained by a study of the protein binding of iodotyrosines. Additionalinformation on serum hormonal iodine purification and quantitative estimation by Sephadex filtration is described. METHODS
AND
MATERIALS
Filtration on Sephadex G-25 (fine) was carried out in 2.5 x 17 cm columns as previously described’. Equilibration with 0.2 M ammonium acetate pH 5.80 or with 0.01 N NaOH was done a few hours before pouring the gel into the column. Flow-rate was 40 ml/h. Fractions of 2 ml were collected. The distribution of free and proteinbound radioactive di-iodotyrosine was studied using charcoal adsorption of the free amino acid. Radioactive iodine-labeled DIT in a volume of 0.0~0 ml was added to 5 ml of pooled normal human serum. After centrifugation at 2000 xg for IO min, 2 ml of the supernatant were pipetted off for radioactivity measurement. Control experiments in the absence of serum showed that nearly 99.9% of radioactive DIT was bound to the charcoal. Adjustment of serum samples to the desired pH was performed at room temperature by addition of I N HCl or 5 N NaOH and continually monitored by a pH-meter. W-labeled thyroglobulin was purified from the thyroid gland of rats which received a tracer dose of lzsI (50 ,uC) as NaI, 24 h before sacrifice. Sucrose gradient centrifugation of the 105000 xg supernatant of thyroid gland homogenate in 0.1 M phosphate buffer pH 6.8 was carried out in the SW 25 rotor of a Spinco I-2 ultrato the 19 S peak centrifuge as previously describeda. The fractions corresponding were used. Concentration of Sephadex fractions containing serum proteins and iodocompounds was performed by centrifugation in a Centriflo membrane cone ultrafilter (Amicon, The Hague, Netherlands) as described by the manufacturer. Half volume reduction was obtained in about 2 h. Purification of radioactive iodinated amino acids was performed by Sephadex G-25 filtration on 2.5 x 17 cm columns in 0.2 M ammonium acetate (iodotyrosines) or in 0.01 N NaOH (T4 or T,). Aliquots of fractions at the peak maximum were used for supplementing serum samples. In all experiments carried out to study DIT serum protein binding VCYSUSDIT concentration, the same amount of (W)DIT was used for each DIT concentration. Before gel filtration or electrophoresis, serum samples were incubated with radioactive iodine-labeled iodoamino acids at 37’ for 30 min. Radioactive iodine countings were carried out in a (71in. Chim. Acta, 3.5 (1971) 28jprg8
BLOOD
IODOCO~~POUNDS
SEPHADEX
287
FILTRATIOh’
Packard Autogamma spectrometer. Activated charcoal was purchased from Merck (Darmstadt, Germany).1311- or 1251-labeled DIT or MIT (specific radioactivity, 25 to 74 mC/mg), 13rI- or lz51-labeled T* or T3 (specific radioactivity, 25 to 75 mC/mg) carrier free hTa+f and 1311-labeled human serum albumin (30 ~C/mg) were purchased from the Commissariat & 1’Energie Atomique (Saclay, France). Purified human serum prealbumin and rabbit antiserum to human serum were purchased from Boehringer (Mannheim, Germany). RESULTS
Aliquots of pooled human serum (PBI = 6 to 8 ,ug I/IOO ml) were adjusted to the desiredpH between 5 and IO and supplemented with 12SI-labeled DIT (0.002 ,ug/ml serum). The distribution of radioactive DIT between free (adsorbed to charcoal) and protein-bound forms was examined using the charcoal method. Fig. I: shows that the fraction of radioactive DIT bound to serum protein increases from pH 6 to pH IO. At the same DIT concentration, serum dilution decreases protein binding (Fig:. IB) indicating that DIT binding is governed by a reversible equilibrium. At a given pH
4
5
6
7
8
9
10
PH Fig, T. DIT binding to human serum protein Z)WS’SUS pH. A, undiluted serum; B, serum diluted (I : I) with 0.2 M ammonium acetate, tris-Cl or tris to obtain the required PH. Analysis by the charcoal method. zoooo counts~min [1e61]DIT and 0.004 ,lg DITjml.
/
0.f ADDED
0.5 DIT lgg/mt serum)
1 )
Fig. 2. DIT binding to human serum protein vwsus DIT concentration charcoal method; 12000 counts/min [1251]DIT per ml serum.
at pH
8.2.
Clie. Chim. Ada, 35
Analysis by the
(1971)
285--298
288
RISM UTH f?t d.
TABLE
I
DIT BIN~IP~GTO SERUM PROTEIN V~YSWS DIT ____._ .._ _-.
COXCENTRATIOK
AND
pH
Serum
PH
EFFLUENT lmlf
EFFLUENT (ml)
l;ig. 3. Sephades G-25 gel filtration of human serum supplenlcnted with an equal amount of [i’JjI]DIT. The column was equilibrated at pH S.6 or at pH 7.4 in 0.05 M tris-Cl. Fig. 1. Effect of DIT concentration on the elution volume of protein-bound [iz51]DIT. Gel filtration of 3-1111human serum sample on Sephadex G-15 equilibrated in 0.01 M NaOH. I, 0.024 pg DIT/roo ml serum; 2, 0.95 pg/roo ml; 3, IOO pg/roo ml; 4. IOOO/A~~/IOO ml.
DIT
Pff
UINIlISG
TO
SERUM
Protein “’ .0
PROTEIN
USING
bound DIT
SEPHADEX
_ _____.---.
Filfratim
G-25
GEL
FILTRATION
AT
DIFFERENT
pHs
is2
0.2 M ammonium acetate 0.05 M tris-Cl 0.05 M t&-Cl 0.01 ILPNaOH -..-_ [1251]DIT was added to a concentration of 0.2~ ~~/IOO ml serum. 3 ml of pooled human serum Iabclcd with about 2oooo countsjmin [1251]DI’lY.
5.8
7-4 8.6 rr.7
0. I
20. I 66.0 19.2
Gin. Chim. ;9cta, 35 (1971) 285-298
BLOOD IODOCOMPOUNDS SEPHADEX FILTRATION
recovered brating
in the void volume is dependent
the gel (Fig. 3 and Table
At pH 5.80 only about 0.1%
II).
upon the pH of the buffer used for equili-
Maximum
is recovered
289
DIT
associated
binding is observed
at pH 8.6.
with proteins.
Serial experiments on the relation between the degree of protein binding and serum DIT concentration were carried out by Sephadex G-25 filtration in 0.01 N NaOH. These conditions of gel filtration were found important as a step in the quantitative estimation of serum iodocompounds (see below). Table III shows that the percent of DIT bound to protein increases with increasing amount of DIT added to serum up to I pg/roo ml and is essentially linearly related to the DIT concentration (from I to 50 pg/roo ml serum). In these conditions, a precise estimation of proteinbound DIT WYSUSfree DIT is difficult to obtain, since free DIT is eluted from the gel in the same elution volume as iodide, i.e., immediately after the void volume. The peak containing free DIT and iodide tends to overlap the protein peak when high amounts of DIT are added to the serum. However these experiments showed an TABLE
III
DIT BINDING AT pH 11.7 DIT
added
#ug/Ioo
mlserum
TO SERUM
PROTEIN
DIT
ZIeYSUS
DIT
CONCENTRATION
/lug/IO0 mlserum
0.02
‘5.1
0.003
0.047
20.4
0.008
36.6 13.1 15.6 18.3 13.2
0.34 0.66 1.56 3.66 6.60
5.0 IO.0 20.0
50.0
Three ml of pooled human serum labeled with 3500 counts/min
Fig. 5. (0) or massie tris-Cl serum
SEPHADEX
GEL
FILTRATION
bound to swuwz protein
%
1.0
USING
[1251]DIT.
gels after electrophoresis in the absence Distribution of [ 1251]DIT in polyacrylamide-agarose in the presence of human serum ( l) Below, photograph of the gel after staining with Cooblue and desiccation. A, albumin; PA, prealbumin. Tris-EDTA-borate buffer pH 8.35 or pH 8.6; migration for 2 h at 180 V. Note that the DIT peak in the presence or absence of migrates far ahead (6.5 cm) of the prealbumin band (5.4 cm). Clin. Chinz.
Acta, 35
(1971)
285-298
BISMUTH
290
et a/.
additional phenomenon. At very low DIT concentrations, DIT is eluted from the gel in a fraction of the protein peak which was slightly retarded (Fig. 4). As the DIT concentration increases the radioactive DIT peak shifts towards the protein absorbance peak and coincides with it at a concentration of IOOO pg/~oo ml serum. This may suggest the presence in human serum of two different DIT-binding proteins each with different binding affinity and capacity. All attempts to show DIT protein binding by zone electrophoresis have failed. In all the systems tested [125I]DIT migrates at the same position in the presence of absence of serum: in the prealbumin zone in paper electrophoresis in glycineeacetate buffer pH 8.6 (ref. 9), ahead of the prealbumin in acrylamide-agarose1° in tris-EDTA-borate buffer pH 8.35 (Fig. 5) or in the albumin zone on cellulose acetate in barbital buffers at pH 5.0, 7.4 or 8.6. In contradiction with the results of Ross and Tapleys we have been unable to show any displacement of [1311]T4 from thyroxine-binding prealbumin by DIT (up to an excess of 40 moles of DIT per mole of T,) using either whole human serum or purified prealbumin. The concentration of [~1251]T,used was 2.2 pug per ml of human serum or per ml of human prealbumin (I mg/ml) However, T, added (5 to IO pg/ml) to human serum labeled with [1251]DIT displaces protein-bound DIT as shown by Sephadex gel filtration at pH 7.4 or by the charcoal method at pHs 7.4 and 8.6 (unpublished observations). Double diffusion in agar of human serum supplemented with radioactive DIT against anti-human serum rabbit antiserum failed to show a radioactive precipitin arc(s) as is clearly demonstrated when radioactive T, replaces redioactive DIT. The same negative results are obtained by immunoelectrophoresis in agarose and diffusion against anti-human serum rabbit antiserum. It is therefore probable that the dissociation constant of the DIT-serum protein complex is so high that the bonds between protein and ligand are broken during electrophoresis. Double jiltration of semrn OTZ Sephadex G-2 5 Separation by gel filtration in dilute NaOH of total serum T4 and T3 from iodide and other iodocompounds has been well documented 4~11*12.Fig. 6 shows the results of a typical experiment performed using Sephadex G-25 equilibrated in 0.01 N NaOH to separate [‘““IIT, and [‘““IIT, from iodide and [1311]thyroglobulin. 2.
EFFLUENT Iml) Fig. 6. Sephadex G-25 gel filtration of j ml of human serum supplemented and [1311]rat thyroglobulin. Column was equilibrated with 0.01 N NaOH. Clin. Chim. Acta, 35 (1971) 285-198
with [‘2bI]‘I-,,
[‘-IIT,
BLOOD
IODOCOMPOUNDS
SEPHADEX
FILTRATION
1 IHSA orT+
EFFLUENT (ml) Fig. 7. Sephadex G-25 gel filtration of 3 ml human serum supplemented with [12sI]DIT and [1311]T,, or [1311]human serum albumin. Column was equilibrated with 0.2 M ammonium acetate pH 5.80. The dotted line indicates the elution volume of MIT as shown in another experiment.
Previous studies6 and the results of the experiments related above show that Sephadex G-25 filtration at pH 5.80 separates tota DIT from MIT as well as from iodide, leaving unresolved the iodocompounds eluting in the void volume (iodothyronines bound to protein and iodoproteins). Fig. 7 illustrates these findings. It is therefore apparent that a single filtration on gel does not permit the separation of the iodocompounds that may be present in serum. Complete separation was obtained by a technique of consecutive filtration at pH 5.8 and pH 11.7 using a single sample of serum. A 3-ml sample of human serum was supplemented with [1251]DIT, [lz51]T4 and [1311]thyroglobulin or [1311]human serum albumin and filtered through a Sephadex G-25 column (2.5 x 17 cm) equilibrated with 0.2 M ammonium acetate, pH 5.80. The excluded or PBI peak contains T4 and iodinated proteins whereas iodide and free DIT are well separated (Fig. 8). Other experiments showed that T, behaved as T4 and that MIT eluted between iodide and DIT. When purified iodo-compounds were used, the recovery for each of them was nearly quantitative (99.8 to 100%). The fractions forming the excluded peak which contained iodothyronines and iodinated proteins (IO to 12 ml) were pooled and concentrated either by centrifugation in membrane cone filters to a volume of 3-4 ml or by the following procedure. The pooled fractions were adjusted to pH 11.7 with 5 N NaOH and layered on the top of a stopped Sephadex G-25 column equilibrated with 0.01 N NaOH; 3 g of dry Sephadex G-25 were then
40
80
120
160
EFFLUENT (ml) Fig. 8. Same experiment as in Fig. j but human serum was supplemented with [‘251]DIT, T, and [‘3*I]human serum albumin. In AA (arrow) the buffer was changed to tertiary saturated with 2 N NH,OH. O, lzjI; l , 1311. Cli+z. Chiwz. Acta,
[*251]amylol
35 (1971) 285-298
BISMUTH
292 added and g that obtained. 100%
standing
for min, the was eluted 0.01 N separation of from T., T,, not recoveries of the iodocompounds to serum 100%). Calculations based on experiments using varying in range 0.002 0.2 ,ug/ml.
a
(98.5
of labeled T, respectively.
as the
EFFLUENT
iodoamino
acids
99.85
to
and 98.5
et d. Fig. is
and 99.0%
(ml)
Fig. 9. Sephadex gel filtration in 0.01 N NaOH of the radioactive peak excluded from the gel in the experiment of Fig. 8. Fractions IZ to 15 were pooled adjusted to pH 11.0, added on the top of the column and 2 g of dry Sephadex G-z5 were added, followed by elution with 0.01 N NaOH. c:, lzsI; l , 1311. Final recovery in duplicate experiments of [12jI]T, and [la111human serum albumin was 98.~7~ and 98.1 :/o respectively.
3. Apfdication to the study of DIT metabolism in man We have shown that filtration of serum on Sephadex
G-25 at pH 5.80 traced
with [W]DIT and supplemented with stable DIT in the range 0.02 to IOO ,ug/roo ml allows 99.85 to gg.oo”/O recovery of total DIT as the free amino acid. Only 0.10 to 0.15% of the radioactivity remains bound to serum protein. Although Sephadexpurified labeled DIT was used in these experiments, the probability that it might contain a small amount of impurity capable of protein binding cannot be eliminated. This could account for the small radioactive fraction recovered in the protein peak. The fact that the percent of radioactivity eluting in the protein peak does not change with increasing concentrations of total DIT added to serum confirms this hypothesis. No additional data are available to demonstrate this possibility. However, since 99.9% of total DIT can be recovered as free DIT after filtration at pH 5.80 it is obvious that the method should be of importance in evaluating the total amount of circulating iodotyrosines in normal and pathologic conditions. Experiments on plasma DIT clearance in normal human subjects favours this statement. Two euthyroid subjects were given orally L-T, (150 ,ug daily) for 5 days to lower thyroid activity. On the sixth day they were given intravenously [lZ51]DIT (20 or 23.75 mg of [1271]DIT traced with Sephadex-purified [W]DIT, 2 &/mg total DIT) in a volume of 4 to 5 ml. Blood samples were obtained at 5 min, 2,6,24 and 48 h after injection and the serum was analyzed by Sephadex filtration at pH 5.80. Table Clin. Chim.
Acta,
35 (1971) 285-29X
RLOOD IODOCOMPOIJNDS SEPHADEX FILTRATION
293
IV shows the distribution of lz61 in the protein, iodide, NIT and DIT peaks. The transient appearance of labeled MIT, the rapid disappearance of labeled DIT and its absence 24 h after [1Z51]DIT injection are obvious. If the radioactivity in the protein peak corresponded to DIT, one can calculate the increase in protein-bound lz71 it should provoke. For all times studied the estimated PBlz71 is always lower than the calculated one and returns to the level at zero time or to a lower value 48 h after DIT administration. It is therefore clear that no free or protein-bound DIT remains in the blood 48 h after the injection of about 20 mg of DIT, thus demonstrating that complete deiodination and excretion has occurred.
TIME COURSE
OF CLEARANCE
OF [iY]DIT
countslminl
3 ml No.
I.
FROM THE BLOOD OF TWO ~~~~~~~~~~
% of total serum vadioactioity
/.q/IOO
ml
HUMAN
EUTHYROID
pglIo0
SUBJECTS
ml
GIA.. cl 0.08 % 24 48
No. 2. CBS..
13163 4 297 2 392 825 I45
2.2 6.3 9.5 17.0
3.2 68.4 84.1 83.0
I.2 o o o
56.5
43.5
0
2.3 63.7 85.9
I.1 0 o
91.1 66.6
o 0
93.4 25.3 6.4 o
5.0 4-I 3.5 2.0
0
1.4
95.0 32.3 6.7
2.1 2.2 2.1
o 0
1.4 1.5
8.7 7.8 7.2 5.7 5.1
0 0.08 2
6 24 48
7054 3 591 1785 983 329
=,7 4.0 7.4 8.9 33.4
3.7 4.4 6.2 6.1 5.0 3.2
5.6
a Subject I received 23,75 mg DIT labeled with 3.32 x IO’ counts/min
7.7 7.8 7.7 7.0 7.1
[lZSI]DIT and subject
5.6
::rl 6.1 2.1
2 20 mg DIT
labeled with 2.80 x IO’ counts/nun [i251]DIT. b Assuming that the radioactivity represents DTT. e Zero time estimated PBi271 + calculated extra-iz71. Three ml pH 5.80. the ratio Duplicate
of serum were filtered on a 2.5 x 17 cm Sephadex G-25 column equilibrated in 0.2 M ammonium acetate, The fractions corresponding to the protein peak were pooled, I271 determination was performed and i2’I/A 280 calculated and related to TOOml serum. All I271 estimations were performed in duplicate. values did not differ by more than so/i,
When commercial preparations of [12SI]DIT (0.2 mC and 6.9 ,ug 13’1) were supplemented with z ml human serum and filtered on a Sephadex G-25 column (2.5 x 17 cm) at pH 5.80, 0.34% of the radioactivity (590000 counts/min) was recovered in the PBlz51 peak (IO ml). Two ml (about I x 10~ counts/min) of the latter after the addition of 3 pg [1271]DIT were refiltered on Sephadex at the same pH; 50 to 60% of the initial radioactivity of the PBW was associated with the excluded fraction*. If it were bound DIT it should correspond to a concentration of 12’Eof about IO ,~g/roo ml. This is not the case since no 1271or an amount too low to be estimated was detected in the excluded fraction. * The remainder of the radioactivity was recovered in the iodide volume of elution and as a trailing zone between iodide and DIT peaks. C&z. Chint. A&a, 35 (xg7r) 285-298
BISMUTH et a!.
294
Fifty percent of the protein-bound radioactivity was not extractable by fsbutanol at pH I. After extraction with n-butanol at pH I, the non-extractable iodinated serum protein material was dialyzed overnight against 0.2 M ammonium bicarbonate buffer, pH 8.0 and digested with pronase at 37” for 24 h with an enzyme to protein ratio of I : m. After acidification to pH 5.80 and filtration on Sephadex at pH 5.80, the bulk of the radioactivity was recovered in two heterogeneous peaks eluting between the void volume and the volume of elution of DIT. These most likely represent iodinated peptides. No discrete peaks of MIT and DIT were observed. These experiments lend support to the idea that some contaminating non-DIT material contained in the / W]DIT preparation reacts covalently with serum protein. This material could still contaminate Sep~adex-puri~ed labeled DIT, since after refiltration at pH 5.80 in the presence of serum, 0.08 to 0.1% of the radioactivity is still present in the protein peak. This was independent of the absolute concentration of [1271]D1T added (up to 1.5 pgjml serum). ~~t~~at~5~~of ~~0~~0~~~~ ~Q~~~~fT4+ TJ by S~~~.a~~.~ G-25 gd ~~t~at~5~~ Using previous observations of Mougey and Masona that T4 was dissociated from its complexes with tllyroxine-binding proteins in dilute NaOH, Jones and Shultzrl described a method of Sephadex filtration allowing total recovery of T, from serum. The method consisted of allowing a serum sample to penetrate into a Sephadex column equihbrated with 0.015 N NaOH, washing extensively with 0.025 M tris-Cl pH 8.6 and eluting T4 with 2 N NH,OH. The method was shown to be of interest in obtaining T* free of contaminating iodinated radiographic contrast agents. However, the volume of N&OH necessary to recover T, of the serum sample is high (45 ml) and the total recovery does not exceed about 8ooj, as shown by tracing with labeled T,. About 18% was lost in the tris washing volume. We have modified this technique to obtain an improved yield of T4 which in our conditions approaches gq.8 I 0.1%. Three-ml serum samples were applied to a 2.5 x rg-cm Sephadex G-e5 column equilibrated with o.015 1v NaOH; 0.025 M tris-Cl pH 7.4 (120 ml) was used to wash the column instead of using the same buffer at pH 8.6. This procedure suppresses the trailing which follows the elution of iodide. Elution of Sephadex-bound Tg was performed using the same buffer containing 0.5% (w/v) SDS. The first 20 ml of eluent after shifting to the eluting buffer was discarded and the next ~~-25 ml collected and dried in wcuo in the presence
EFFLUENT (ml) Fig. IO. Sephadex G-25 gel filtration of human serum (3 ml) incubated with [131I]T, using the canditions described in the text. Arrow, cha,nge of buffer to the same buffer containing 0.5% SDS. Non-purified [1311]T4 added, 0.025 pg (specific radioactivity, ro.9 mC/mg). C&k ChfW.. AC&Z,35 (X971)
285-298
BLOOD IOD~~OMPOUNDS SEPHADES
FILTRATION
29.5
of P,O,. The residue was taken up in 3 ml of water and its iodine content estimated in a Technicon AutoAnalyzer equipped with an automatic digester. A typical filtration using trace amounts of [1311]T, as a marker is shown in Fig. IO. In 18 column runs the recovery of radioactive thyroxine in the final eluate amounted to 99.8o/0 f 0.2. The same results were obtained using T, and a mixture of T,+T,. Tables V to VI+VII show that I. the presence of SDS does not interfere with 127I determination, and 2. the recovery of T, from the vessel used to concentrate the eluate is obtained with an accuracy of about f 1% (Table VII). Using this technique, the results of hormonal iodine estimation in several lots of pooled human serum from euthyroid or hyperthyroid subjects are shown in Table VIII. These results are compared to the PB12’1 and hormonal iodine determination using the resin method of Backer et ~1.~~. TABLE
V
INFLIJENCEOF SDS ygjroo
ON
“‘1
ESTIMATIOS
ml
-_ latI added=
1571 estimated 5.0 9.6
5.0
10.0 15.0
14.8
20.0
19.6
a 127I as KIO, was dissolved in o.0~5 M tris-Cl pH 7.4 containing TAI3LE
VI
INFLUENCE
OF INCREASING
CONCENTRATIONS
OF
SDS
lz71 estimated in solutions
concentration
containing
%
5 %uglIOOmE
IO pgj+oo
0.5 1.5
5.0 5.0 4.9 4,9 5.0 5,1
10.0 10.0 9.9
j.1
10.0
2.0
2.5 3.0 3.5 1~0
RECOVERY
OF THYROXINE
AFTER
30
increasing con-
Rad~oac~~u~~y counts/g min 53.110
‘5
in 0.0~5 M tris-Cl pH 7.4 containing
DESICCATION
53.830
20
mt
10.0
2 10
lz71 ESTIMATION
10.0 IO.0
0
5
ON
VII
Stable tkyy~oxine added f%
SDS
-~
ml) was dissolved KIO, (j OTT IOp,g l*‘I/roo centrations of SDS (w/v). TABLE
0.5% (w/v) SDS.
52.300 52.340 53.390 52.470 53.190
T, was dissolved in 25 ml 0.025 M tris-Cl pH 7.4 containing residue dissolved in 3 ml water.
0.5%
SDS (w/v) desiccated
i%Z. Chim. Acta, 35
(1971)
and the
285-298
BISiWJTH
296
et d.
As previously shown by Jones and Shultzlr the method is useful in eliminating the interference of some iodinated radiographic agents with the quantitative estimation of hormonal iodine (Table VIII). TABLE
VIII
,Vatuve of S~YUWZ
iodine
PBI
Hormonal
,jcg ‘2’I/IOO
Sqbhadex method pg 1271/I00 ml
Krszn methoda
4.5 i 0.1 4.8 * 0.j 8.1 * 0.1 IZ.2 4.9
3.8 3.6 Y.2 I‘+.8 >3o >30 5.7 5 ‘2 5.9
ml
_.
Normal” Normal” Normalb Hyperthyroid Serum after aortography
5.5 * o.Xe 5.4 * 0.x 12.4 $- 0.6 16.0 >3o
Normal h id. -+ Contrix” Normal B id. + Contrix”
6.2 >30 7.6 & 1.2 >3o
j.8
a b C * e
(108 g/ml) (336 g/ml)
j.2
5.5 4.7 5 0.8 4.4 i 0.8
Racker et al., ref. 13. Pooled human serum. Injection of Contrix. Contrix added in vitro. The column was washed with 250 ml 0.025 M tris-Cl pH 7.4. Mean + standard deviation.
DISCUSSION
Van
Zyl
and Wilson14
demonstrated
by protein
precipitation
and dialysis
experiments that MIT and DIT when present in serum are bound to serum proteins. However, other investigators 15*16found, that the iodotyrosines in serum were more than 90% dialyzable. Up to now, no unequivocal demonstration of iodotyrosine binding to serum protein by electrophoretic methods has been demonstrated. Alpers et al.17 briefly mentioned that in human serum supplemented with [1311]DIT, DIT migrated in association with serum albumin using paper electrophoresis (barbital buffer pH 8.6) ; no experimental details and no control of free DIT migration were given. Block et al. I8 fractionated serum protein by DEAE cellulose chromatography after addition to the serum of a pancreatin digest of 1311-labeled thyroid extract. They found that [131I]DIT was mainly recovered in the elution volume of the gamma globulins. However a control experiment without added serum was not performed. This control using the conditions of stepwise elution as proposed by Peterson and Sober19 might show that free DIT would be eluted with the same buffer that eluted gamma globulins. When labeled DIT was incubated with human serum prior to paper electrophoresis, DIT migrated with the prealbumin or the albumin fractions when trismaleate buffer, pH 8.4 or barbital buffer, pH 8.6 were used respectively14. We have not been able to confirm these findings. Ross and Tapleys reported that DIT could displace T4 bound to serum prealbumin using paper eletrophoresis at pH 8.6 in glycine-acetate buffer. However we were not able to reproduce their experiments even using a purified prealbumin and a 4o-fold molar excess of DIT over T4. Recently, Clin. Chim. Acta,
35 (1971)
285-298
BLOOD
IODOCOMPOUNDS
Hao and Tabachnick20, ineffective
in displacing
SEPHADEX
FILTRATION
using starch gel electrophoresis
297 at pH 8.6, found that DIT was
[1251]T, from purified th~oxine-binding
globulin.
All our attempts to show DIT binding to serum proteins by zone electrophoretic methods using various migration media or by immunological double diffusion in agar were unsuccessful. It is therefore misleading to calculate’ that in terms of DIT binding the maximal capacity of serum would be 10x-172 pg/~oo ml, assuming from the results of Ross and TapleyO a similar binding capacity of prealbumin for T, and DIT. However, methods using Sephadex filtration or adsorption of free DIT on charcoal demonstrate that serum proteins are able to bind DIT with a very low affinity since electrophoretic forces apparently break the bonds linking protein to l&and. Di-iodotyrosine binding to serum protein depends on pH and protein concentration. This indicates that DIT binding is governed by a reversible equilibrium. Below pH 6.0 the fraction of protein-bound DIT, as shown by Sephadex filtration at pH $30, corresponds to only 0.10 to 0.15% of the total added radioactive DIT. Although purified [lZ51]DIT was used in these experiments, it is not unlikely that it may contain a very low amount of a non-DIT radioactive contaminant which could remain proteinbound at pH 5.80 and would account for the O.IO-0.15% fraction recovered in the serum protein peak after Sephadex filtration, This suggestion is confirmed by the data reported in the fourth paragraph of the RESULTS section. In 1963, we concluded6 from experiments performed by Sephadex filtration at pH 5.80 that iodotyrosines were absent from the serum of rats in isotopic equilibrium with 125I and receiving daily 5 to 50 pg of ls71 as iodide. Later Zeidler and GraulZ1, using Sephadex filtration at pH 7.4 of serum supplemented with jZ3*IjDIT or obtained after in viva injection of [ 1311]DIT, argued that iodotvrosines, if present in the circulation, would not be free but would be protein bound. They concluded that gel filtration was not useful for serum iodotyrosine determination. This is obviously a misleading statement, since our previous experiments were carried out at pH 5.80 and, as shown in this paper, more than g$3°/o of the serum DIT is recovered by filtration as the free iodoamino acid. Therefore, the results of our previous investigations on the absence of iodotyrosines in the serum of normal rats remain fully pertinent to further discussion on the questionable problem of circulating iodotyrosines. It is estimated by some investigators that the concentration of iodotyrosines in human blood is roughly in the range of 1-3 pg1100 ml serum7. This is not supported by our experiments on the DIT clearance from the blood of T,-blocked euthyroid humans. Twenty-four hours after a single injection of about 20 mg of [1251]DIT, no free MIT or DIT was detected in the serum as shown by Sephadex filtration at pH 5.80 and no increase in the PB1z71 corresponding to the radioactivity associated with the protein peak was observed. It is therefore most unlikely that some DIT reversibly bound to protein could remain and accumulate in the blood. This would be contradictory to the assumption of a reversible equilibrium between protein-bound and free DIT. The conflicting results reported in the literature on the presence or absence of iodotyrosines in the blood of euthyroid mammals and man should be explained either by methodological uncertainties or by the presence of iodotyrosine-like compounds or iodotyrosine degradation products covalently linked to serum proteins. This will be discussed later in another communication The method of double filtration on Sephadex described in this paper for the C&a. Chim.Acta,35
(1971) 285-298
BISMUTH et cd.
298
separation of all the known serum iodo-compounds was shown to be accurate and reproducible. It gives almost quantjtative results when reasonable amounts of radioactivity are present in the serum. Attempts to estimate lZ71in the separated fractions are encouraging. Kriiskemper and KGddinglZ briefly reported the use of filtration on Sephadex and a stepwise elution with sodium acetate and 0.01 N NaOH to separate contaminating iodinated radiographic contrast agents (Urografin, Biligrafin) from iodotyrosines, iodothyronines and iodoproteins but no quantitative data were given. Finally, improvement of the method described by Jones and Shultz” for the separation and quantitative estimationof total T, --i--T,from the serum was found reproducible and useful when determination of T, by competitive binding analysis is not available. REFERENCES I 2 3 4 5 6
7 8
9 ID II 12 13 14 15 16 17 18
S. LISSITZKY, J. BISE*IUTHAXD M. ROLLAND, C~,~n,C~~~.~c~a, 7 (1962) 183. H. SPITZY, H. SKRUBE AXD K. MUELLER, Mikrochim. Acta, 2 (1961) 296. S. L~ssr~zituAND J. BISMUTH,C&Z.C~~~. A&, 8(1963) 269. E. II.MOUGEY AND J. W. MASON, Awl. Biochem., 6(1963) 223. E. MAKOWETZ, K. M~~LLERAND H. SPITZY, iMicvachem.T.,10(x966) 194. S. LISSITZKY, J. BISMUTH AND C. SIMON, Nature, x99 (i963) IOOZ. B. A. RIIODES, Acta Endocrinol., .Su~~l. 127 (1968) 57. S. LISSITZKY, M. ROQUES, J. TORRESANI AND C. SIMON,BUL Sot. Chim. Biol., 47 (1965) 1999. J. E. Ross AND D. F. TAPLEY, Endocrinology, 79 (1966) 493. J, URI~L, Bull. Sac. Chim. Biol., 48 (1966) 969. J. E. JONES AND J. S. SHULTZ, J. Clin.Endocrinol.,27 (1967) X77. H.L. KR~~SKEMPER AKD R. KSDDING, Klin. Woc/wchr.,46(196S) 143. E. T. BACRER,T.J. POSTMES AND J. D. WIEXER,C&Z. Chim. Acta, 15 (1967) 77. A. \‘AX 2% AND B. WILSOX, S. African J. Lab. CEin. Med., IO (1964) 15. W. TOXG, A. TAUROG AND I.L. CKAIKOFF,.[.B~O~. Chem., 207 (1954) 59. R. M. RLIZZAR~ ANII J. NIOSIER,,4.M. A. J. L)iseasrs Ch~~d~p~z,94 (1957) 534. J. B. ALPERS, M. L. PETERMANN AND J. E. RALL, Arch. Biochem. B~opk~s.~ 65 (1956) 513. R. J. B~oce, R. H. MANDL, S. KELLER AND S.C. WEREER, Arcb.Bioche~.Biopk~s., 7j (1958)
19 E. A. PETERSON AND H. A. SOBER, J. Aww. Chevn. Sot., 78 (1956) 20 Y. L. HACI AND M. TABACHNICK, Endocrinology,SX (1971) 81. 21 LT. _&IDLER AXD E. H. GRAUL, Nature, 206 (1965) 401,
Clin.CAim. Acta,
35 (1971)
285-298
757.
CCA
CHIMICA
285
ACTA
4512
SEPHADEX BINDING
JANINE
FILTRATION
BISMUTH,
Laboratoive de Biochimie Sante’ et de la Rechevche rpMavseille 5 (France) (Received
OF SERUM
IODOCOMPOUNDS
AND
PROTEIN-
OF IODOTYROSINES”
April
MATHILDE Mkdicale Mgdicale,
CASTAY
AND SERGE
et Unite’ thyroi’dienne Faculte’ de Me’decine.
LISSITZKY
de l’lnstitut National 2 7 Bd. Jealz-Moulin,
de la
8, 1971)
SUMMARY
Di-iodotyrosine binding to serum protein cannot be demonstrated either by electrophoresis on paper, cellulose acetate or acrylamide-agarose (using various buffers at different pHs) or by double diffusion in agar and immunoelectrophoresis. To the contrary, binding of DIT to serum protein can be estimated by Sephadex G-25 gel filtration and by adsorption on charcoal. Binding increases with pH between 6 and IO. It is almost negligible below pH 6. This property was used to propose a method of separation of all the known iodocompounds found in serum using a single serum sample and two consecutive filtrations on Sephadex G-25. The first filtration at pH 5.80 allows the separation of MIT from DIT and iodide and from PBI. The latter is resolved into T4, T, and iodoprotein by refiltration in 0.01 N NaOH. Recoveries of the radioactive iodine-labeled iodocompounds are almost quantitative. Sephadex gel filtration is useful in estimating the iodotyrosine content of serum. Experiments on DIT clearance from the blood in man support the idea that iodotyrosines are not normal components of the blood of euthyroid humans. The usefulness of Sephadex G-25 gel filtration in 0.01 N NaOH for the quantitative estimation of serum total hormonal iodine is confirmed.
Gel filtration on Sephadex at a pH near neutrality has been used to separate iodide from iodothyronines bound to serum proteins as well as from free iodothyronines1-3. Further studies showed that when equilibrated in diluted NaOH the method allowed to separate T4 and T, from iodide4-5. In these experimental conditions free iodotyrosines are eluted along with iodide whereas iodinated proteins, when present, are recovered in the void volume of the column. Sephadex filtration at pH 5.80 (ref. 6) separated DIT from MIT and iodide but left unresolved the iodine-containing material * This study was supported by the Commissariat & 1’Energie Atomique the Centre National de la Recherche Scientifique (Paris, France). Abbreviations: MIT, 3.iodotyrosine; DIT, thyroxine; SDS, sodium dodecylsulphate.
x,5-di-iodotyrosine;
T,,
(Saclay,
France)
x,5,3’-tri-iodothyronine;
Clin. Chin<. Acta,
35 (1971)
and by T,,
285-298
286
BISMUTH et cd.
excluded from the gel which contains protein-bound iodothyronines proteins. Serum protein binding of iodotyrosines, if any, can interfere
and iodinated with the inter-
pretation of the results of gel filtration methods used to resolve serum iodinated compounds. No systematic and conclusive investigation has been performed on the distribution of free and protein-bound serum iodotyrosines (see ref. 7 for a review). Study of the distribution of iodo-compounds in the blood is of fundamental importance for the diagnosis of thyroid diseases in man. The need of a simple, accurate and reproducible method allowing the separation of all the known serum iodocompounds is therefore obvious. In the present communication we describe a method of separation of the presently known iodocompounds by filtration on Sephadex G-25 equilibrated at two different pHs and using a single serum sample. The validity of the method has been ascertained by a study of the protein binding of iodotyrosines. Additionalinformation on serum hormonal iodine purification and quantitative estimation by Sephadex filtration is described. METHODS
AND
MATERIALS
Filtration on Sephadex G-25 (fine) was carried out in 2.5 x 17 cm columns as previously described’. Equilibration with 0.2 M ammonium acetate pH 5.80 or with 0.01 N NaOH was done a few hours before pouring the gel into the column. Flow-rate was 40 ml/h. Fractions of 2 ml were collected. The distribution of free and proteinbound radioactive di-iodotyrosine was studied using charcoal adsorption of the free amino acid. Radioactive iodine-labeled DIT in a volume of 0.0~0 ml was added to 5 ml of pooled normal human serum. After centrifugation at 2000 xg for IO min, 2 ml of the supernatant were pipetted off for radioactivity measurement. Control experiments in the absence of serum showed that nearly 99.9% of radioactive DIT was bound to the charcoal. Adjustment of serum samples to the desired pH was performed at room temperature by addition of I N HCl or 5 N NaOH and continually monitored by a pH-meter. W-labeled thyroglobulin was purified from the thyroid gland of rats which received a tracer dose of lzsI (50 ,uC) as NaI, 24 h before sacrifice. Sucrose gradient centrifugation of the 105000 xg supernatant of thyroid gland homogenate in 0.1 M phosphate buffer pH 6.8 was carried out in the SW 25 rotor of a Spinco I-2 ultrato the 19 S peak centrifuge as previously describeda. The fractions corresponding were used. Concentration of Sephadex fractions containing serum proteins and iodocompounds was performed by centrifugation in a Centriflo membrane cone ultrafilter (Amicon, The Hague, Netherlands) as described by the manufacturer. Half volume reduction was obtained in about 2 h. Purification of radioactive iodinated amino acids was performed by Sephadex G-25 filtration on 2.5 x 17 cm columns in 0.2 M ammonium acetate (iodotyrosines) or in 0.01 N NaOH (T4 or T,). Aliquots of fractions at the peak maximum were used for supplementing serum samples. In all experiments carried out to study DIT serum protein binding VCYSUSDIT concentration, the same amount of (W)DIT was used for each DIT concentration. Before gel filtration or electrophoresis, serum samples were incubated with radioactive iodine-labeled iodoamino acids at 37’ for 30 min. Radioactive iodine countings were carried out in a (71in. Chim. Acta, 3.5 (1971) 28jprg8
BLOOD
IODOCO~~POUNDS
SEPHADEX
287
FILTRATIOh’
Packard Autogamma spectrometer. Activated charcoal was purchased from Merck (Darmstadt, Germany).1311- or 1251-labeled DIT or MIT (specific radioactivity, 25 to 74 mC/mg), 13rI- or lz51-labeled T* or T3 (specific radioactivity, 25 to 75 mC/mg) carrier free hTa+f and 1311-labeled human serum albumin (30 ~C/mg) were purchased from the Commissariat & 1’Energie Atomique (Saclay, France). Purified human serum prealbumin and rabbit antiserum to human serum were purchased from Boehringer (Mannheim, Germany). RESULTS
Aliquots of pooled human serum (PBI = 6 to 8 ,ug I/IOO ml) were adjusted to the desiredpH between 5 and IO and supplemented with 12SI-labeled DIT (0.002 ,ug/ml serum). The distribution of radioactive DIT between free (adsorbed to charcoal) and protein-bound forms was examined using the charcoal method. Fig. I: shows that the fraction of radioactive DIT bound to serum protein increases from pH 6 to pH IO. At the same DIT concentration, serum dilution decreases protein binding (Fig:. IB) indicating that DIT binding is governed by a reversible equilibrium. At a given pH
4
5
6
7
8
9
10
PH Fig, T. DIT binding to human serum protein Z)WS’SUS pH. A, undiluted serum; B, serum diluted (I : I) with 0.2 M ammonium acetate, tris-Cl or tris to obtain the required PH. Analysis by the charcoal method. zoooo counts~min [1e61]DIT and 0.004 ,lg DITjml.
/
0.f ADDED
0.5 DIT lgg/mt serum)
1 )
Fig. 2. DIT binding to human serum protein vwsus DIT concentration charcoal method; 12000 counts/min [1251]DIT per ml serum.
at pH
8.2.
Clie. Chim. Ada, 35
Analysis by the
(1971)
285--298
288
RISM UTH f?t d.
TABLE
I
DIT BIN~IP~GTO SERUM PROTEIN V~YSWS DIT ____._ .._ _-.
COXCENTRATIOK
AND
pH
Serum
PH
EFFLUENT lmlf
EFFLUENT (ml)
l;ig. 3. Sephades G-25 gel filtration of human serum supplenlcnted with an equal amount of [i’JjI]DIT. The column was equilibrated at pH S.6 or at pH 7.4 in 0.05 M tris-Cl. Fig. 1. Effect of DIT concentration on the elution volume of protein-bound [iz51]DIT. Gel filtration of 3-1111human serum sample on Sephadex G-15 equilibrated in 0.01 M NaOH. I, 0.024 pg DIT/roo ml serum; 2, 0.95 pg/roo ml; 3, IOO pg/roo ml; 4. IOOO/A~~/IOO ml.
DIT
Pff
UINIlISG
TO
SERUM
Protein “’ .0
PROTEIN
USING
bound DIT
SEPHADEX
_ _____.---.
Filfratim
G-25
GEL
FILTRATION
AT
DIFFERENT
pHs
is2
0.2 M ammonium acetate 0.05 M tris-Cl 0.05 M t&-Cl 0.01 ILPNaOH -..-_ [1251]DIT was added to a concentration of 0.2~ ~~/IOO ml serum. 3 ml of pooled human serum Iabclcd with about 2oooo countsjmin [1251]DI’lY.
5.8
7-4 8.6 rr.7
0. I
20. I 66.0 19.2
Gin. Chim. ;9cta, 35 (1971) 285-298
BLOOD IODOCOMPOUNDS SEPHADEX FILTRATION
recovered brating
in the void volume is dependent
the gel (Fig. 3 and Table
At pH 5.80 only about 0.1%
II).
upon the pH of the buffer used for equili-
Maximum
is recovered
289
DIT
associated
binding is observed
at pH 8.6.
with proteins.
Serial experiments on the relation between the degree of protein binding and serum DIT concentration were carried out by Sephadex G-25 filtration in 0.01 N NaOH. These conditions of gel filtration were found important as a step in the quantitative estimation of serum iodocompounds (see below). Table III shows that the percent of DIT bound to protein increases with increasing amount of DIT added to serum up to I pg/roo ml and is essentially linearly related to the DIT concentration (from I to 50 pg/roo ml serum). In these conditions, a precise estimation of proteinbound DIT WYSUSfree DIT is difficult to obtain, since free DIT is eluted from the gel in the same elution volume as iodide, i.e., immediately after the void volume. The peak containing free DIT and iodide tends to overlap the protein peak when high amounts of DIT are added to the serum. However these experiments showed an TABLE
III
DIT BINDING AT pH 11.7 DIT
added
#ug/Ioo
mlserum
TO SERUM
PROTEIN
DIT
ZIeYSUS
DIT
CONCENTRATION
/lug/IO0 mlserum
0.02
‘5.1
0.003
0.047
20.4
0.008
36.6 13.1 15.6 18.3 13.2
0.34 0.66 1.56 3.66 6.60
5.0 IO.0 20.0
50.0
Three ml of pooled human serum labeled with 3500 counts/min
Fig. 5. (0) or massie tris-Cl serum
SEPHADEX
GEL
FILTRATION
bound to swuwz protein
%
1.0
USING
[1251]DIT.
gels after electrophoresis in the absence Distribution of [ 1251]DIT in polyacrylamide-agarose in the presence of human serum ( l) Below, photograph of the gel after staining with Cooblue and desiccation. A, albumin; PA, prealbumin. Tris-EDTA-borate buffer pH 8.35 or pH 8.6; migration for 2 h at 180 V. Note that the DIT peak in the presence or absence of migrates far ahead (6.5 cm) of the prealbumin band (5.4 cm). Clin. Chinz.
Acta, 35
(1971)
285-298
BISMUTH
290
et a/.
additional phenomenon. At very low DIT concentrations, DIT is eluted from the gel in a fraction of the protein peak which was slightly retarded (Fig. 4). As the DIT concentration increases the radioactive DIT peak shifts towards the protein absorbance peak and coincides with it at a concentration of IOOO pg/~oo ml serum. This may suggest the presence in human serum of two different DIT-binding proteins each with different binding affinity and capacity. All attempts to show DIT protein binding by zone electrophoresis have failed. In all the systems tested [125I]DIT migrates at the same position in the presence of absence of serum: in the prealbumin zone in paper electrophoresis in glycineeacetate buffer pH 8.6 (ref. 9), ahead of the prealbumin in acrylamide-agarose1° in tris-EDTA-borate buffer pH 8.35 (Fig. 5) or in the albumin zone on cellulose acetate in barbital buffers at pH 5.0, 7.4 or 8.6. In contradiction with the results of Ross and Tapleys we have been unable to show any displacement of [1311]T4 from thyroxine-binding prealbumin by DIT (up to an excess of 40 moles of DIT per mole of T,) using either whole human serum or purified prealbumin. The concentration of [~1251]T,used was 2.2 pug per ml of human serum or per ml of human prealbumin (I mg/ml) However, T, added (5 to IO pg/ml) to human serum labeled with [1251]DIT displaces protein-bound DIT as shown by Sephadex gel filtration at pH 7.4 or by the charcoal method at pHs 7.4 and 8.6 (unpublished observations). Double diffusion in agar of human serum supplemented with radioactive DIT against anti-human serum rabbit antiserum failed to show a radioactive precipitin arc(s) as is clearly demonstrated when radioactive T, replaces redioactive DIT. The same negative results are obtained by immunoelectrophoresis in agarose and diffusion against anti-human serum rabbit antiserum. It is therefore probable that the dissociation constant of the DIT-serum protein complex is so high that the bonds between protein and ligand are broken during electrophoresis. Double jiltration of semrn OTZ Sephadex G-2 5 Separation by gel filtration in dilute NaOH of total serum T4 and T3 from iodide and other iodocompounds has been well documented 4~11*12.Fig. 6 shows the results of a typical experiment performed using Sephadex G-25 equilibrated in 0.01 N NaOH to separate [‘““IIT, and [‘““IIT, from iodide and [1311]thyroglobulin. 2.
EFFLUENT Iml) Fig. 6. Sephadex G-25 gel filtration of j ml of human serum supplemented and [1311]rat thyroglobulin. Column was equilibrated with 0.01 N NaOH. Clin. Chim. Acta, 35 (1971) 285-198
with [‘2bI]‘I-,,
[‘-IIT,
BLOOD
IODOCOMPOUNDS
SEPHADEX
FILTRATION
1 IHSA orT+
EFFLUENT (ml) Fig. 7. Sephadex G-25 gel filtration of 3 ml human serum supplemented with [12sI]DIT and [1311]T,, or [1311]human serum albumin. Column was equilibrated with 0.2 M ammonium acetate pH 5.80. The dotted line indicates the elution volume of MIT as shown in another experiment.
Previous studies6 and the results of the experiments related above show that Sephadex G-25 filtration at pH 5.80 separates tota DIT from MIT as well as from iodide, leaving unresolved the iodocompounds eluting in the void volume (iodothyronines bound to protein and iodoproteins). Fig. 7 illustrates these findings. It is therefore apparent that a single filtration on gel does not permit the separation of the iodocompounds that may be present in serum. Complete separation was obtained by a technique of consecutive filtration at pH 5.8 and pH 11.7 using a single sample of serum. A 3-ml sample of human serum was supplemented with [1251]DIT, [lz51]T4 and [1311]thyroglobulin or [1311]human serum albumin and filtered through a Sephadex G-25 column (2.5 x 17 cm) equilibrated with 0.2 M ammonium acetate, pH 5.80. The excluded or PBI peak contains T4 and iodinated proteins whereas iodide and free DIT are well separated (Fig. 8). Other experiments showed that T, behaved as T4 and that MIT eluted between iodide and DIT. When purified iodo-compounds were used, the recovery for each of them was nearly quantitative (99.8 to 100%). The fractions forming the excluded peak which contained iodothyronines and iodinated proteins (IO to 12 ml) were pooled and concentrated either by centrifugation in membrane cone filters to a volume of 3-4 ml or by the following procedure. The pooled fractions were adjusted to pH 11.7 with 5 N NaOH and layered on the top of a stopped Sephadex G-25 column equilibrated with 0.01 N NaOH; 3 g of dry Sephadex G-25 were then
40
80
120
160
EFFLUENT (ml) Fig. 8. Same experiment as in Fig. j but human serum was supplemented with [‘251]DIT, T, and [‘3*I]human serum albumin. In AA (arrow) the buffer was changed to tertiary saturated with 2 N NH,OH. O, lzjI; l , 1311. Cli+z. Chiwz. Acta,
[*251]amylol
35 (1971) 285-298
BISMUTH
292 added and g that obtained. 100%
standing
for min, the was eluted 0.01 N separation of from T., T,, not recoveries of the iodocompounds to serum 100%). Calculations based on experiments using varying in range 0.002 0.2 ,ug/ml.
a
(98.5
of labeled T, respectively.
as the
EFFLUENT
iodoamino
acids
99.85
to
and 98.5
et d. Fig. is
and 99.0%
(ml)
Fig. 9. Sephadex gel filtration in 0.01 N NaOH of the radioactive peak excluded from the gel in the experiment of Fig. 8. Fractions IZ to 15 were pooled adjusted to pH 11.0, added on the top of the column and 2 g of dry Sephadex G-z5 were added, followed by elution with 0.01 N NaOH. c:, lzsI; l , 1311. Final recovery in duplicate experiments of [12jI]T, and [la111human serum albumin was 98.~7~ and 98.1 :/o respectively.
3. Apfdication to the study of DIT metabolism in man We have shown that filtration of serum on Sephadex
G-25 at pH 5.80 traced
with [W]DIT and supplemented with stable DIT in the range 0.02 to IOO ,ug/roo ml allows 99.85 to gg.oo”/O recovery of total DIT as the free amino acid. Only 0.10 to 0.15% of the radioactivity remains bound to serum protein. Although Sephadexpurified labeled DIT was used in these experiments, the probability that it might contain a small amount of impurity capable of protein binding cannot be eliminated. This could account for the small radioactive fraction recovered in the protein peak. The fact that the percent of radioactivity eluting in the protein peak does not change with increasing concentrations of total DIT added to serum confirms this hypothesis. No additional data are available to demonstrate this possibility. However, since 99.9% of total DIT can be recovered as free DIT after filtration at pH 5.80 it is obvious that the method should be of importance in evaluating the total amount of circulating iodotyrosines in normal and pathologic conditions. Experiments on plasma DIT clearance in normal human subjects favours this statement. Two euthyroid subjects were given orally L-T, (150 ,ug daily) for 5 days to lower thyroid activity. On the sixth day they were given intravenously [lZ51]DIT (20 or 23.75 mg of [1271]DIT traced with Sephadex-purified [W]DIT, 2 &/mg total DIT) in a volume of 4 to 5 ml. Blood samples were obtained at 5 min, 2,6,24 and 48 h after injection and the serum was analyzed by Sephadex filtration at pH 5.80. Table Clin. Chim.
Acta,
35 (1971) 285-29X
RLOOD IODOCOMPOIJNDS SEPHADEX FILTRATION
293
IV shows the distribution of lz61 in the protein, iodide, NIT and DIT peaks. The transient appearance of labeled MIT, the rapid disappearance of labeled DIT and its absence 24 h after [1Z51]DIT injection are obvious. If the radioactivity in the protein peak corresponded to DIT, one can calculate the increase in protein-bound lz71 it should provoke. For all times studied the estimated PBlz71 is always lower than the calculated one and returns to the level at zero time or to a lower value 48 h after DIT administration. It is therefore clear that no free or protein-bound DIT remains in the blood 48 h after the injection of about 20 mg of DIT, thus demonstrating that complete deiodination and excretion has occurred.
TIME COURSE
OF CLEARANCE
OF [iY]DIT
countslminl
3 ml No.
I.
FROM THE BLOOD OF TWO ~~~~~~~~~~
% of total serum vadioactioity
/.q/IOO
ml
HUMAN
EUTHYROID
pglIo0
SUBJECTS
ml
GIA.. cl 0.08 % 24 48
No. 2. CBS..
13163 4 297 2 392 825 I45
2.2 6.3 9.5 17.0
3.2 68.4 84.1 83.0
I.2 o o o
56.5
43.5
0
2.3 63.7 85.9
I.1 0 o
91.1 66.6
o 0
93.4 25.3 6.4 o
5.0 4-I 3.5 2.0
0
1.4
95.0 32.3 6.7
2.1 2.2 2.1
o 0
1.4 1.5
8.7 7.8 7.2 5.7 5.1
0 0.08 2
6 24 48
7054 3 591 1785 983 329
=,7 4.0 7.4 8.9 33.4
3.7 4.4 6.2 6.1 5.0 3.2
5.6
a Subject I received 23,75 mg DIT labeled with 3.32 x IO’ counts/min
7.7 7.8 7.7 7.0 7.1
[lZSI]DIT and subject
5.6
::rl 6.1 2.1
2 20 mg DIT
labeled with 2.80 x IO’ counts/nun [i251]DIT. b Assuming that the radioactivity represents DTT. e Zero time estimated PBi271 + calculated extra-iz71. Three ml pH 5.80. the ratio Duplicate
of serum were filtered on a 2.5 x 17 cm Sephadex G-25 column equilibrated in 0.2 M ammonium acetate, The fractions corresponding to the protein peak were pooled, I271 determination was performed and i2’I/A 280 calculated and related to TOOml serum. All I271 estimations were performed in duplicate. values did not differ by more than so/i,
When commercial preparations of [12SI]DIT (0.2 mC and 6.9 ,ug 13’1) were supplemented with z ml human serum and filtered on a Sephadex G-25 column (2.5 x 17 cm) at pH 5.80, 0.34% of the radioactivity (590000 counts/min) was recovered in the PBlz51 peak (IO ml). Two ml (about I x 10~ counts/min) of the latter after the addition of 3 pg [1271]DIT were refiltered on Sephadex at the same pH; 50 to 60% of the initial radioactivity of the PBW was associated with the excluded fraction*. If it were bound DIT it should correspond to a concentration of 12’Eof about IO ,~g/roo ml. This is not the case since no 1271or an amount too low to be estimated was detected in the excluded fraction. * The remainder of the radioactivity was recovered in the iodide volume of elution and as a trailing zone between iodide and DIT peaks. C&z. Chint. A&a, 35 (xg7r) 285-298
BISMUTH et a!.
294
Fifty percent of the protein-bound radioactivity was not extractable by fsbutanol at pH I. After extraction with n-butanol at pH I, the non-extractable iodinated serum protein material was dialyzed overnight against 0.2 M ammonium bicarbonate buffer, pH 8.0 and digested with pronase at 37” for 24 h with an enzyme to protein ratio of I : m. After acidification to pH 5.80 and filtration on Sephadex at pH 5.80, the bulk of the radioactivity was recovered in two heterogeneous peaks eluting between the void volume and the volume of elution of DIT. These most likely represent iodinated peptides. No discrete peaks of MIT and DIT were observed. These experiments lend support to the idea that some contaminating non-DIT material contained in the / W]DIT preparation reacts covalently with serum protein. This material could still contaminate Sep~adex-puri~ed labeled DIT, since after refiltration at pH 5.80 in the presence of serum, 0.08 to 0.1% of the radioactivity is still present in the protein peak. This was independent of the absolute concentration of [1271]D1T added (up to 1.5 pgjml serum). ~~t~~at~5~~of ~~0~~0~~~~ ~Q~~~~fT4+ TJ by S~~~.a~~.~ G-25 gd ~~t~at~5~~ Using previous observations of Mougey and Masona that T4 was dissociated from its complexes with tllyroxine-binding proteins in dilute NaOH, Jones and Shultzrl described a method of Sephadex filtration allowing total recovery of T, from serum. The method consisted of allowing a serum sample to penetrate into a Sephadex column equihbrated with 0.015 N NaOH, washing extensively with 0.025 M tris-Cl pH 8.6 and eluting T4 with 2 N NH,OH. The method was shown to be of interest in obtaining T* free of contaminating iodinated radiographic contrast agents. However, the volume of N&OH necessary to recover T, of the serum sample is high (45 ml) and the total recovery does not exceed about 8ooj, as shown by tracing with labeled T,. About 18% was lost in the tris washing volume. We have modified this technique to obtain an improved yield of T4 which in our conditions approaches gq.8 I 0.1%. Three-ml serum samples were applied to a 2.5 x rg-cm Sephadex G-e5 column equilibrated with o.015 1v NaOH; 0.025 M tris-Cl pH 7.4 (120 ml) was used to wash the column instead of using the same buffer at pH 8.6. This procedure suppresses the trailing which follows the elution of iodide. Elution of Sephadex-bound Tg was performed using the same buffer containing 0.5% (w/v) SDS. The first 20 ml of eluent after shifting to the eluting buffer was discarded and the next ~~-25 ml collected and dried in wcuo in the presence
EFFLUENT (ml) Fig. IO. Sephadex G-25 gel filtration of human serum (3 ml) incubated with [131I]T, using the canditions described in the text. Arrow, cha,nge of buffer to the same buffer containing 0.5% SDS. Non-purified [1311]T4 added, 0.025 pg (specific radioactivity, ro.9 mC/mg). C&k ChfW.. AC&Z,35 (X971)
285-298
BLOOD IOD~~OMPOUNDS SEPHADES
FILTRATION
29.5
of P,O,. The residue was taken up in 3 ml of water and its iodine content estimated in a Technicon AutoAnalyzer equipped with an automatic digester. A typical filtration using trace amounts of [1311]T, as a marker is shown in Fig. IO. In 18 column runs the recovery of radioactive thyroxine in the final eluate amounted to 99.8o/0 f 0.2. The same results were obtained using T, and a mixture of T,+T,. Tables V to VI+VII show that I. the presence of SDS does not interfere with 127I determination, and 2. the recovery of T, from the vessel used to concentrate the eluate is obtained with an accuracy of about f 1% (Table VII). Using this technique, the results of hormonal iodine estimation in several lots of pooled human serum from euthyroid or hyperthyroid subjects are shown in Table VIII. These results are compared to the PB12’1 and hormonal iodine determination using the resin method of Backer et ~1.~~. TABLE
V
INFLIJENCEOF SDS ygjroo
ON
“‘1
ESTIMATIOS
ml
-_ latI added=
1571 estimated 5.0 9.6
5.0
10.0 15.0
14.8
20.0
19.6
a 127I as KIO, was dissolved in o.0~5 M tris-Cl pH 7.4 containing TAI3LE
VI
INFLUENCE
OF INCREASING
CONCENTRATIONS
OF
SDS
lz71 estimated in solutions
concentration
containing
%
5 %uglIOOmE
IO pgj+oo
0.5 1.5
5.0 5.0 4.9 4,9 5.0 5,1
10.0 10.0 9.9
j.1
10.0
2.0
2.5 3.0 3.5 1~0
RECOVERY
OF THYROXINE
AFTER
30
increasing con-
Rad~oac~~u~~y counts/g min 53.110
‘5
in 0.0~5 M tris-Cl pH 7.4 containing
DESICCATION
53.830
20
mt
10.0
2 10
lz71 ESTIMATION
10.0 IO.0
0
5
ON
VII
Stable tkyy~oxine added f%
SDS
-~
ml) was dissolved KIO, (j OTT IOp,g l*‘I/roo centrations of SDS (w/v). TABLE
0.5% (w/v) SDS.
52.300 52.340 53.390 52.470 53.190
T, was dissolved in 25 ml 0.025 M tris-Cl pH 7.4 containing residue dissolved in 3 ml water.
0.5%
SDS (w/v) desiccated
i%Z. Chim. Acta, 35
(1971)
and the
285-298
BISiWJTH
296
et d.
As previously shown by Jones and Shultzlr the method is useful in eliminating the interference of some iodinated radiographic agents with the quantitative estimation of hormonal iodine (Table VIII). TABLE
VIII
,Vatuve of S~YUWZ
iodine
PBI
Hormonal
,jcg ‘2’I/IOO
Sqbhadex method pg 1271/I00 ml
Krszn methoda
4.5 i 0.1 4.8 * 0.j 8.1 * 0.1 IZ.2 4.9
3.8 3.6 Y.2 I‘+.8 >3o >30 5.7 5 ‘2 5.9
ml
_.
Normal” Normal” Normalb Hyperthyroid Serum after aortography
5.5 * o.Xe 5.4 * 0.x 12.4 $- 0.6 16.0 >3o
Normal h id. -+ Contrix” Normal B id. + Contrix”
6.2 >30 7.6 & 1.2 >3o
j.8
a b C * e
(108 g/ml) (336 g/ml)
j.2
5.5 4.7 5 0.8 4.4 i 0.8
Racker et al., ref. 13. Pooled human serum. Injection of Contrix. Contrix added in vitro. The column was washed with 250 ml 0.025 M tris-Cl pH 7.4. Mean + standard deviation.
DISCUSSION
Van
Zyl
and Wilson14
demonstrated
by protein
precipitation
and dialysis
experiments that MIT and DIT when present in serum are bound to serum proteins. However, other investigators 15*16found, that the iodotyrosines in serum were more than 90% dialyzable. Up to now, no unequivocal demonstration of iodotyrosine binding to serum protein by electrophoretic methods has been demonstrated. Alpers et al.17 briefly mentioned that in human serum supplemented with [1311]DIT, DIT migrated in association with serum albumin using paper electrophoresis (barbital buffer pH 8.6) ; no experimental details and no control of free DIT migration were given. Block et al. I8 fractionated serum protein by DEAE cellulose chromatography after addition to the serum of a pancreatin digest of 1311-labeled thyroid extract. They found that [131I]DIT was mainly recovered in the elution volume of the gamma globulins. However a control experiment without added serum was not performed. This control using the conditions of stepwise elution as proposed by Peterson and Sober19 might show that free DIT would be eluted with the same buffer that eluted gamma globulins. When labeled DIT was incubated with human serum prior to paper electrophoresis, DIT migrated with the prealbumin or the albumin fractions when trismaleate buffer, pH 8.4 or barbital buffer, pH 8.6 were used respectively14. We have not been able to confirm these findings. Ross and Tapleys reported that DIT could displace T4 bound to serum prealbumin using paper eletrophoresis at pH 8.6 in glycine-acetate buffer. However we were not able to reproduce their experiments even using a purified prealbumin and a 4o-fold molar excess of DIT over T4. Recently, Clin. Chim. Acta,
35 (1971)
285-298
BLOOD
IODOCOMPOUNDS
Hao and Tabachnick20, ineffective
in displacing
SEPHADEX
FILTRATION
using starch gel electrophoresis
297 at pH 8.6, found that DIT was
[1251]T, from purified th~oxine-binding
globulin.
All our attempts to show DIT binding to serum proteins by zone electrophoretic methods using various migration media or by immunological double diffusion in agar were unsuccessful. It is therefore misleading to calculate’ that in terms of DIT binding the maximal capacity of serum would be 10x-172 pg/~oo ml, assuming from the results of Ross and TapleyO a similar binding capacity of prealbumin for T, and DIT. However, methods using Sephadex filtration or adsorption of free DIT on charcoal demonstrate that serum proteins are able to bind DIT with a very low affinity since electrophoretic forces apparently break the bonds linking protein to l&and. Di-iodotyrosine binding to serum protein depends on pH and protein concentration. This indicates that DIT binding is governed by a reversible equilibrium. Below pH 6.0 the fraction of protein-bound DIT, as shown by Sephadex filtration at pH $30, corresponds to only 0.10 to 0.15% of the total added radioactive DIT. Although purified [lZ51]DIT was used in these experiments, it is not unlikely that it may contain a very low amount of a non-DIT radioactive contaminant which could remain proteinbound at pH 5.80 and would account for the O.IO-0.15% fraction recovered in the serum protein peak after Sephadex filtration, This suggestion is confirmed by the data reported in the fourth paragraph of the RESULTS section. In 1963, we concluded6 from experiments performed by Sephadex filtration at pH 5.80 that iodotyrosines were absent from the serum of rats in isotopic equilibrium with 125I and receiving daily 5 to 50 pg of ls71 as iodide. Later Zeidler and GraulZ1, using Sephadex filtration at pH 7.4 of serum supplemented with jZ3*IjDIT or obtained after in viva injection of [ 1311]DIT, argued that iodotvrosines, if present in the circulation, would not be free but would be protein bound. They concluded that gel filtration was not useful for serum iodotyrosine determination. This is obviously a misleading statement, since our previous experiments were carried out at pH 5.80 and, as shown in this paper, more than g$3°/o of the serum DIT is recovered by filtration as the free iodoamino acid. Therefore, the results of our previous investigations on the absence of iodotyrosines in the serum of normal rats remain fully pertinent to further discussion on the questionable problem of circulating iodotyrosines. It is estimated by some investigators that the concentration of iodotyrosines in human blood is roughly in the range of 1-3 pg1100 ml serum7. This is not supported by our experiments on the DIT clearance from the blood of T,-blocked euthyroid humans. Twenty-four hours after a single injection of about 20 mg of [1251]DIT, no free MIT or DIT was detected in the serum as shown by Sephadex filtration at pH 5.80 and no increase in the PB1z71 corresponding to the radioactivity associated with the protein peak was observed. It is therefore most unlikely that some DIT reversibly bound to protein could remain and accumulate in the blood. This would be contradictory to the assumption of a reversible equilibrium between protein-bound and free DIT. The conflicting results reported in the literature on the presence or absence of iodotyrosines in the blood of euthyroid mammals and man should be explained either by methodological uncertainties or by the presence of iodotyrosine-like compounds or iodotyrosine degradation products covalently linked to serum proteins. This will be discussed later in another communication The method of double filtration on Sephadex described in this paper for the C&a. Chim.Acta,35
(1971) 285-298
BISMUTH et cd.
298
separation of all the known serum iodo-compounds was shown to be accurate and reproducible. It gives almost quantjtative results when reasonable amounts of radioactivity are present in the serum. Attempts to estimate lZ71in the separated fractions are encouraging. Kriiskemper and KGddinglZ briefly reported the use of filtration on Sephadex and a stepwise elution with sodium acetate and 0.01 N NaOH to separate contaminating iodinated radiographic contrast agents (Urografin, Biligrafin) from iodotyrosines, iodothyronines and iodoproteins but no quantitative data were given. Finally, improvement of the method described by Jones and Shultz” for the separation and quantitative estimationof total T, --i--T,from the serum was found reproducible and useful when determination of T, by competitive binding analysis is not available. REFERENCES I 2 3 4 5 6
7 8
9 ID II 12 13 14 15 16 17 18
S. LISSITZKY, J. BISE*IUTHAXD M. ROLLAND, C~,~n,C~~~.~c~a, 7 (1962) 183. H. SPITZY, H. SKRUBE AXD K. MUELLER, Mikrochim. Acta, 2 (1961) 296. S. L~ssr~zituAND J. BISMUTH,C&Z.C~~~. A&, 8(1963) 269. E. II.MOUGEY AND J. W. MASON, Awl. Biochem., 6(1963) 223. E. MAKOWETZ, K. M~~LLERAND H. SPITZY, iMicvachem.T.,10(x966) 194. S. LISSITZKY, J. BISMUTH AND C. SIMON, Nature, x99 (i963) IOOZ. B. A. RIIODES, Acta Endocrinol., .Su~~l. 127 (1968) 57. S. LISSITZKY, M. ROQUES, J. TORRESANI AND C. SIMON,BUL Sot. Chim. Biol., 47 (1965) 1999. J. E. Ross AND D. F. TAPLEY, Endocrinology, 79 (1966) 493. J, URI~L, Bull. Sac. Chim. Biol., 48 (1966) 969. J. E. JONES AND J. S. SHULTZ, J. Clin.Endocrinol.,27 (1967) X77. H.L. KR~~SKEMPER AKD R. KSDDING, Klin. Woc/wchr.,46(196S) 143. E. T. BACRER,T.J. POSTMES AND J. D. WIEXER,C&Z. Chim. Acta, 15 (1967) 77. A. \‘AX 2% AND B. WILSOX, S. African J. Lab. CEin. Med., IO (1964) 15. W. TOXG, A. TAUROG AND I.L. CKAIKOFF,.[.B~O~. Chem., 207 (1954) 59. R. M. RLIZZAR~ ANII J. NIOSIER,,4.M. A. J. L)iseasrs Ch~~d~p~z,94 (1957) 534. J. B. ALPERS, M. L. PETERMANN AND J. E. RALL, Arch. Biochem. B~opk~s.~ 65 (1956) 513. R. J. B~oce, R. H. MANDL, S. KELLER AND S.C. WEREER, Arcb.Bioche~.Biopk~s., 7j (1958)
19 E. A. PETERSON AND H. A. SOBER, J. Aww. Chevn. Sot., 78 (1956) 20 Y. L. HACI AND M. TABACHNICK, Endocrinology,SX (1971) 81. 21 LT. _&IDLER AXD E. H. GRAUL, Nature, 206 (1965) 401,
Clin.CAim. Acta,
35 (1971)
285-298
757.