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Thyroidal iodinated compounds in nodular goitre
CLINICA
I27
CHIMICA ACTA
THYROIDAL
G. M. LEVIS,
IODINATED
D. A. KOUTRAS,
COMPOUNDS
A. VAGENAKIS,
IN NODULAR
G. MESSARIS,
GOITRE
C. MIRAS
AND
B. MALAMOS
Athens University, Department of Clinical Therapeutics, Alexandra Hospital, T/as. Sofas and Lourou Str., Athens 61r (Greece) (Received
December
12,
1967)
SUMMARY
The iodinated
compounds
in the nodular
and paranodular
tissues of rg cases
of nodular goitre have been separated by thin-layer chromatography. A high monoiodotyrosine/di-iodotyrosine ratio was found in “cold” nodules, and a low one in “hot” nodules.
Two unknown
iodinated
compounds
have been detected
in the nodular and
paranodular tissue in some cases, and evidence is presented suggesting that these are iodinated lipids. Their possible role in diverting iodine from the normal metabolic pathway
is discussed.
INTRODUCTION
The biochemistry
of thyroid
tissue is by now well studied,
and the main iodi-
nated compounds detected are 3-mono-iodo-tyrosine (MIT), x,5-di-iodo-tyrosine 3,5,3’,5’-l-tetra-iodo-thyronine (thyroxine, T.J and 3,5,x’-l-tri-iodo-thyronine
(DIT), (T3)1--3.
In cases of nodular non-toxic goitre previous investigations have revealed abnormalities of the composition of the iodinated thyroid compounds, consisting mainly in low concentrations of iodothyronines (T3, T4) and an abnormally high MIT/DIT ratio4@. The present study has confirmed these findings, and has shown in addition the presence in certain cases of two ioclinated compounds with a precise mobility on chromatograms of the hydrolysate, which have been identified as iodinated lipids. MATERIALS
AND METHODS
Surgical thyroid gland specimens from rg patients subjected to total or partial thyroidectomy were studied. Diagnosis was based on the usual clinical and radioisotopic criteriaa, including in every case a dot scan and a photo-scan of the thyroid gland. One to seven clays prior to surgery, 250 ,I_& of lz61 was administered to the patients orally. Immediately after surgery, a part of both nodular and paranodular tissues was kept for pathological study. Similar parts were immediately processed for biochemical analysis or kept at -20~. Homogenates of the tissue were prepared in g volumes of saline solution in a Waring blendor at 2’-4’. The homogenate was brought Clin.
Chim.
Acta, 20 (1968)
127-134
to pH 8.5 and hydrolysed with trypsin (Difco x : 250) 5 mg per mt for 72 b at 37” in the presence of toluene. The pR was readjusted, when needed, and 2.5 mg of trypsin per ml were added after 24 and 48 h. The hydrolysate was acidified and extracted with ?z-butanol saturated with 0.1 N HCl and the extract was then taken down under reduced pressure. The dq residue was dissolved in methanol-ammonia gg : I: v/v and appropriate quantities were used for the detection of the 1261-containing compounds with the aid of thinlayer chromatography and autoradiography, The plates used for thin-layer chromatography were coated with a mixture of silicagel-G g g and of anion exchange resin DEAE cellulose* 6 g, as described previously7. The plates were developed in two dimensions ~-butanol-acetic acid-water 78 : 5 : 17 v/v and ?s-butanol saturated with ammonia 2 N. Autoradio~~aphy was performed by direct contact, on Kodak Royal Blue X ray films. Identification of spots was performed by simultaneous cbromato~ra~hy on each side of the plate of standardT,, T3, DET, MIT and I-. The areas on the plate corresponding to the radioactive spots were located by depicting the spots of the X-ray film on a piece of paper and producing a matrix out of it. The radioactive spots were then scrubbed out from the plate and counted in a well type scintillation counter (Nuclear Chicago Model C-120-1). For the extraction of lipids, the methanol-ammonia gg: I v/v solution was brought to E ml with water and extracted with 19 ml of ~hloroforl~l-l~ethanol z : I v/v. The non-lipid iodinated amino acids and methanol were then removed into T/S volume of water separated by ~entrif~gation and washed three times with the theoretical upper phase of Folch et al.“. The chloroform phase was concentrated under reduced pressure and ~hromatographed with butanol saturated with ammonia 2 PJ on the same thin-layer plate, with the original butanol extract. The residue of the chloroform phase, containing the lipid material, was also chro~~ato~~aphed on plates coated only with silicagel&, using chloroform-methanol-acetic acid-water 80 : 15 : 3 : z v/v, as developing solvent. A mixture of phospholipids containing : phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin from human leucocytes was simultaneously chromatographed and used as identification standard. Mild alkaline hydrolysis and acid hydrolysis of the lipids were performed as described previouslys. RESULTS
The laboratory and clinical data of the cases investigated as well as the results of ~~lromatogra~hi~ analysis of thyroid tissue are shown in Tables I-III. The pathological and chromato~raphic findings are shown separately for the nodular (N) and the paranodular (I?) tissue. Table I shows the results in II cases of scintigraphically cold nodules. A low percentage of iodothyronines was present at the time of the study, and a high MIT/ DIT ratio in the nodular tissue was evident. Two unknown compounds, designated U, and U, respectively, were present in both the nodular and paranodular tissues of three cases, in the paranodular one of a fourth and the nodular of a fifth case. Contrary to the above results, hot nodules showed a lower MITjDIT ratio in * Diethyla~noethyl cellulose powder for thin-layer New York 6, M.Y., U.S.A. ~&?Z. chi@t. ACta,
20 (1968)
127-K34
chromato~aphy
from Mann Research Lab.,
22
2
;
I
-_--._--_--
Mean
2
2
7
3
7.5
5.5
7.0
6.5
6.8
0.23
0.23
0.10
0.02
0.07
0.06
49.0
55.0
41.6
49.0
53
58
35
-
0.13
-
60
-
-
3
-
0.21
3
3
4
5.8
3
I
6.0
0.07
4
FINDINGS
52.0
6.5
PATHOLOGICAL
0.18
VALUES,
52.0
LABORATORY
TABLE OF NON-TOXIC
“COLD”
N P
N
Solid and microfollicular adenoma Normal tissue ( ?) ~acrofollicular goitre Microfollicular goitre Follicular adenoma Microfollicular goitre Microfollicular adenoma Microfollicular goitre Solid and microfollicular adenoma Microfollicular goitre -
I I CASES
N P N I? N P N P N P N P
IN
Microfollicular adenoma Microfollicular goitre Solid and microfollicular adenoma Colloid goitre Solid and microfollicular adenoma Normal tissue Solid and microfollicular adenoma MicrofolIicular and colloid goitre Microfollicular adenoma Microfollicular and colloid goitre
RESULTS
P N P N P N P N P
AND
31.3 23.2
IO
13 5' 38 24
36 24 54 28
70
25 -
27 19
6
30 27 27 23
14 23
MIT
2.5 36.5
8
31 20
13 38 21 6
55 16
30 39 20
34 33 32
21
zd;
54
42 54
DIT
12.2 _
II.0
77
9
13.2
18
2.5 31
-. -
0.72
0.20
.--
_-
7
-
T,
-
5
18
5
40 4 -
T,
in thvroid
NODULE
lzsI com4ounds
THYROID
-
-
2.8
2.9
7 18
-
II -
22
tissue,
-
-
5.9 7.2
24 -
-
8 29
41 27 7
-
I.96 0.84
I.2
I.2
3.50 0.65 I.50 4.10 0.8 0.6 8 1.2
6.83 0.00
0.59
1.20
0.17
1.10
0.44
I.10
0.55
0.33 0.43
MIT/D11 rafi0
of told
62.0
15
53
‘7
San
19
46
4’
44
16
18
49
/a
3
TABLF:
0’
46.0
‘4
111
61.j
r.3
Mean
68.0
uptake 24 h %
1311
12
.vo.
-
___-
0.44
0.11
0.18
0.05
0.23
0.06
-
--
7.4
-
7.5
5.3
9.0
4.4
0.08
0.96
7.0
Fp*
PBI
0.17
PB’“l/ 48 h %I1
5
L
14
z
2
---
.._ - .._.. _
Cold nodule Cold nodule Cold nodule Hot nodule Cold nodule
adm.
2
nodule Warm nodule
__.-.
3
I
2
Hot
Warm nodule Hot nodule
a&z.
Da)ls after lZSI
______.. -_
PhotoSCalZ
._
P
N P N P N P N P N P
N P
N P N P N I? N P
_~_
__. findings
~_._
._..-
adenoma
Normal tissue Microfollicular
6 41
36 .j8.8
55
45 _~
-
38
61 -
45
44 __
42 -
cyst
Normal tissue Haemorrhagic
_
40.5 34.2
58 44 23 20 35 40 45
DIT
~___ ...___
IS
19
23 16 17 25 jz,
MIT
27 -
Normal tissue Embryonal adenoma Normal tissue
~__
.-___
---_
9.6
43
-
-
.-
---
5 _
to.9
11.7
9 -7 31 ---31 L.
T,
x.4
-
~-
-
-
7 _-
-
-
-~-
T,
--
---
-
-
_
G.6
2.5
-..2 10 -__
U, 2
6 I.84
-
0.9
0.44 I.1
MITIDIT ratio
0.8
-
2
~-
--
c
0.5’ 0.60
1.25 0.00 0.30 0.70
0.51
0.36
0.40
MI T/DIT ratio
oftotal
- .,
4.7 ‘4
6 17 36 -~ _
-
U,
lzaI compounds in thyroid tissue, “d
Solid and lnicrofollicul~r Normal tissue Microfollicular adenoma
atlenoma
--___
Microfollicufar adenoma Mixed micro- and macrofollicular goitre Mixed micro- and macro- fol. adenoma Microfollicular goitre Mixed follicular adenoma Cofloid go&e Mixed follicular adenoma Colloid goitre
Pathological
.__---._
Tissue
._
NODULAR
GOITRE STUDIES
131
the nodular tissue, in spite of the fact that the specimens were examined on an average two days after lz51 administration, against 3 days in the case of the cold nodules. The two unknown compounds were again found in three cases.
a
Fig. I. Autoradiogrsm of thin-layer chromatogram in two dimensions on mixture of Silica gel G and DEAE cellulose. First solvent system n-butanol-acetic acid-water (78: 5: ~7, v/v). Second solvent system n-butanol saturated with 2 -4’ ammonia. Butanol extracts of hydrolysates of thyroid tissue a: case I N; b: case4 P of Table I. CEin. Chim. A&,
20 (1968) 127-134
132
LEVIS
et id.
Table III summarises the results in 5 cases where the paranodular tissue was normal, including case 3 from the cold nodule group. The MIT/DIT ratio was lower than 1.1 in all except the last case, in which the high value found is probably accounted for by the high conversion of DIT to T, and hence the relatively low DIT concentration, The two unknown compounds have not been detected. Representative chromatograms with and without the two unknown compounds are shown in Figs. ra and b. The methanol-ammonia solution of the butanol extract of the hydrolysates of the tissues which contained the unknown spots were extracted with chloroformmethanol as described under METHODS. In all cases the radioactivity of the unknown spots was extractable into chlorofo~. This is shown in Fig. z where both the butano1 extract and the chloroform extract are chromatographed on the same plate. In order to add more information on the nature of the unknown lipids these were subjected to mild alkaline and to acid hydrolysis. It was found that the mobility on thin-layer plates of the two unknown spots was unchanged following mild alkaline hydrolysis but after acid hydrolysis only one spot identical to the iodide was detected. The major radioactive spot of the chloroform extract, when chromatographed on silicagel-G, presented a mobility very similar to that of phosphatidylcholine.
Fig. 2. Autoradiogram of thin-layer chromatogram on mixture of Silica gel G and DEAE) cellulose. Solvent system n-butanol saturated with z N ammonia. A, B, C are butanol extracts of the hydrolysates of cases 14 N, 14 P of Table II and 4 P of Table I. A’, B’, C’ arc the respective chloroform extracts. DISCUSSION
The finding in cold nodules of decreased transfer of iodine from MIT to the more heavily iodinated DIT, and hence a high ~IT~DIT ratio, is not surprising,
IiODULAR
GOITRE
STUDIES
I33
having been previously reported tive stage of iodine metabolism
by others l”$ll. The formation and hence a high MIT/DIT
of MIT is a more primiratio may be found in
several thyroid disorderP : in cold nodules, as here, after goitrogen administration13p1*, in experimental iodine deficiency15v1B, and in spontaneous human iodine deficiency goitrel7. The more active the thyroid, as evident from the 1311 uptake, the more DIT and T, are formed’*. In endemic goitre the low DIT and low iodothyronine concentrations are probably due to the dilution of the thyroidal iodine in a greater tissue mass, since the total iodine content of the gland may be normal, but the iodine concentration per unit mass is reduced16. Furthermore, the studies of Ermans et ~1.~~suggest that this decreased transfer iodothyronines is secondary
of iodine from MIT to DIT and from iodotyrosines to to the goitrous process rather than a primary causative
factor. This is in agreement with the finding by Dimitriadou et aLzo in Thailand that in diffuse goitre of young persons the MIT/DIT ratio is normal, whereas in nodular goitres of older individuals the rate of iodothyronine formation is low, with high MIT/DIT and iodotyrosine/iodothyronine ratios. The finding of unknown iodinated compounds which could be separated by lipid solvents in certain tissues of the examined goitres seems to be interesting. These unknown compounds, besides being lipid soluble, when taken from different subjects, have a definite mobility on thin-layer chromatography, moving faster than T, in the solvent
butanol-ammonia, but not in the solvent front. The occurrence of iodinated lipids has been reported
only after in vitro incu-
bation of 1311 with thyroid cell-free particulate fractions. Thus, De Groot and Carvalhozl suggested the formation of an iodinated lipid compound under the above mentioned experimental conditions, which moved to the solvent front on chromatog, raphy with butanol-ethanol-ammonia. Suzuki et n1.22, under similar conditionsreported the formation of both nonphospholipids and phospholipids which were isolated with chloroform extraction. These compounds, when chromatographed in butanol-dioxaneeammonia, also moved near the solvent front. It must be stressed that inorganic oxidation The lipid-soluble iodinated compounds presented preparations
and specifically
not in the normal
of iodide may produce artifactsz3. here are not found in all thyroid ones, experimentally
treated
in the
same way. In addition, these compounds are found mainly in the paranodular tissue of cold nodule cases. These findings exclude the possibility that unknowns U, and U, are artifacts produced in vitro. The spot usually
found
in larger
quantities
(U,) when chromatographed
in
systems for lipid separation, displays a single spot with mobility similar to phosphatidylcholine. This finding together with the fact that this compound is resistant to mild alcali predicates that the iodide-binding compound is a polar lipid, wherefrom iodide can be removed by acid treatment either through abolishing the binding capacity or through acid hydrolysis of the lipid. Vilkkiz4 has reported that specifically thyroidal lecithin comprises a fraction binding in vitro iodide and this is the conclusion reached by Schneider and WolfP, who also presented evidence suggesting reversible binding of iodide through electrostatic forces. The iodinated lipid compounds described here do not seem to bind iodide reversibly since iodide is not removed during washing of the chloroform phase. It may, therefore, be suggested that the thyroids with the unknown compounds contain increased amounts of such a specific lipid fraction which could bind iodide C&z. Chim. Acta,
20 (1968) 127-134
mms
I34 irreversibly, thus resulting in withdrawal able for thyroid hormone synthesis.
of iodide which, otherwise,
et al.
would be avail-
It is not known whether the formation of iodinated lipids is of primary importance in the aetiology of non-toxic goitre or whether it is secondary to the goitrous process pev se, as suggested by Ermans et al. IQ for the high MIT/DIT ratio. At anp rate, occurring
in a functionally
deficient
gland it would aggravate
the difficulty
in
the biosynthesis of thyroid hormones, diverting iodine from the normal metabolic pathway. Further studies are required in order to elucidate the role of these iodinated compounds in the generation or maintenance of human goitre. ACKNOWLEDGEMEXTS
This work was been supported National
Institutes
of Health,
by Grants No. AM 07464 and AM 08987 from the
Bethesda,
Md., U.S.A.
REFERENCES I R. PITT-RIVERS AND J. E. RALL, Endocrinology, 65 (1961) 309. 2 E. J. WAYNE, D. A. KOUTRAS AND W. I). ALEXANDER, Clivlical Aspcts 0.1Iodine lWetaholism, Blackwell, Oxford, 1964. 3 J. E. RALL, J. ROBBINS AND C. G. LEWALLEN, in G. FI~cus, K. V. THIMANN AND E. B. ASTWOOD (Eds.), The Hormones, vol. V, Academic Press, New York and London, 1964, p. 159. 4 R. PITT-RIVERS, D. HUBBLE AND W. H. HOATHER, J. Clin. Endocrinol., 17 (1957) 1313. 5 A. M. ERMANS, J, E. DUMONT AND P. A. BASTENIE, J. Clin. Endocvinol., 23 (1963) 539. 6 B. K. MALAMOS, G. K. DAIKOS, V. SAMARA AND D. A. KOUTRAS, Acta Endocrinol., 32 (1959) 31, 7 B. K. MALAMOS, J. KIBURIS AND G. LEVIS, journr’es Hell&es de S&wation Imme’diat! et dr Chvomatogvaphie, (IIIt?me J.I.S.I.C.), Association of Greek Chemist, Athens, 1965, p. Irg. 8 J, FOLCH, M. LEES AND S. G. H. SLOANE, ,I. Viol. Chem., 226 (1957) 497. 9 C. MIRAS, J. MANTZOS AND G. LEVIS, Biochen?. J.. 98 (1966) 782. IO G. B. SALABE, S. TONELLI, H. SALABE AND L. BASCHIERI, in C. CASSANO AND M. ANDREOLI (Eds.), Czwvewt Topics in Thvroid Research. Academic Press, New York and London, 1965, p. 900. II H. W. WAHNER, E. GAITAN AND P. CORREA, /. Clin. I
Acta,
20 (1968)
127-134
I27
CHIMICA ACTA
THYROIDAL
G. M. LEVIS,
IODINATED
D. A. KOUTRAS,
COMPOUNDS
A. VAGENAKIS,
IN NODULAR
G. MESSARIS,
GOITRE
C. MIRAS
AND
B. MALAMOS
Athens University, Department of Clinical Therapeutics, Alexandra Hospital, T/as. Sofas and Lourou Str., Athens 61r (Greece) (Received
December
12,
1967)
SUMMARY
The iodinated
compounds
in the nodular
and paranodular
tissues of rg cases
of nodular goitre have been separated by thin-layer chromatography. A high monoiodotyrosine/di-iodotyrosine ratio was found in “cold” nodules, and a low one in “hot” nodules.
Two unknown
iodinated
compounds
have been detected
in the nodular and
paranodular tissue in some cases, and evidence is presented suggesting that these are iodinated lipids. Their possible role in diverting iodine from the normal metabolic pathway
is discussed.
INTRODUCTION
The biochemistry
of thyroid
tissue is by now well studied,
and the main iodi-
nated compounds detected are 3-mono-iodo-tyrosine (MIT), x,5-di-iodo-tyrosine 3,5,3’,5’-l-tetra-iodo-thyronine (thyroxine, T.J and 3,5,x’-l-tri-iodo-thyronine
(DIT), (T3)1--3.
In cases of nodular non-toxic goitre previous investigations have revealed abnormalities of the composition of the iodinated thyroid compounds, consisting mainly in low concentrations of iodothyronines (T3, T4) and an abnormally high MIT/DIT ratio4@. The present study has confirmed these findings, and has shown in addition the presence in certain cases of two ioclinated compounds with a precise mobility on chromatograms of the hydrolysate, which have been identified as iodinated lipids. MATERIALS
AND METHODS
Surgical thyroid gland specimens from rg patients subjected to total or partial thyroidectomy were studied. Diagnosis was based on the usual clinical and radioisotopic criteriaa, including in every case a dot scan and a photo-scan of the thyroid gland. One to seven clays prior to surgery, 250 ,I_& of lz61 was administered to the patients orally. Immediately after surgery, a part of both nodular and paranodular tissues was kept for pathological study. Similar parts were immediately processed for biochemical analysis or kept at -20~. Homogenates of the tissue were prepared in g volumes of saline solution in a Waring blendor at 2’-4’. The homogenate was brought Clin.
Chim.
Acta, 20 (1968)
127-134
to pH 8.5 and hydrolysed with trypsin (Difco x : 250) 5 mg per mt for 72 b at 37” in the presence of toluene. The pR was readjusted, when needed, and 2.5 mg of trypsin per ml were added after 24 and 48 h. The hydrolysate was acidified and extracted with ?z-butanol saturated with 0.1 N HCl and the extract was then taken down under reduced pressure. The dq residue was dissolved in methanol-ammonia gg : I: v/v and appropriate quantities were used for the detection of the 1261-containing compounds with the aid of thinlayer chromatography and autoradiography, The plates used for thin-layer chromatography were coated with a mixture of silicagel-G g g and of anion exchange resin DEAE cellulose* 6 g, as described previously7. The plates were developed in two dimensions ~-butanol-acetic acid-water 78 : 5 : 17 v/v and ?s-butanol saturated with ammonia 2 N. Autoradio~~aphy was performed by direct contact, on Kodak Royal Blue X ray films. Identification of spots was performed by simultaneous cbromato~ra~hy on each side of the plate of standardT,, T3, DET, MIT and I-. The areas on the plate corresponding to the radioactive spots were located by depicting the spots of the X-ray film on a piece of paper and producing a matrix out of it. The radioactive spots were then scrubbed out from the plate and counted in a well type scintillation counter (Nuclear Chicago Model C-120-1). For the extraction of lipids, the methanol-ammonia gg: I v/v solution was brought to E ml with water and extracted with 19 ml of ~hloroforl~l-l~ethanol z : I v/v. The non-lipid iodinated amino acids and methanol were then removed into T/S volume of water separated by ~entrif~gation and washed three times with the theoretical upper phase of Folch et al.“. The chloroform phase was concentrated under reduced pressure and ~hromatographed with butanol saturated with ammonia 2 PJ on the same thin-layer plate, with the original butanol extract. The residue of the chloroform phase, containing the lipid material, was also chro~~ato~~aphed on plates coated only with silicagel&, using chloroform-methanol-acetic acid-water 80 : 15 : 3 : z v/v, as developing solvent. A mixture of phospholipids containing : phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin from human leucocytes was simultaneously chromatographed and used as identification standard. Mild alkaline hydrolysis and acid hydrolysis of the lipids were performed as described previouslys. RESULTS
The laboratory and clinical data of the cases investigated as well as the results of ~~lromatogra~hi~ analysis of thyroid tissue are shown in Tables I-III. The pathological and chromato~raphic findings are shown separately for the nodular (N) and the paranodular (I?) tissue. Table I shows the results in II cases of scintigraphically cold nodules. A low percentage of iodothyronines was present at the time of the study, and a high MIT/ DIT ratio in the nodular tissue was evident. Two unknown compounds, designated U, and U, respectively, were present in both the nodular and paranodular tissues of three cases, in the paranodular one of a fourth and the nodular of a fifth case. Contrary to the above results, hot nodules showed a lower MITjDIT ratio in * Diethyla~noethyl cellulose powder for thin-layer New York 6, M.Y., U.S.A. ~&?Z. chi@t. ACta,
20 (1968)
127-K34
chromato~aphy
from Mann Research Lab.,
22
2
;
I
-_--._--_--
Mean
2
2
7
3
7.5
5.5
7.0
6.5
6.8
0.23
0.23
0.10
0.02
0.07
0.06
49.0
55.0
41.6
49.0
53
58
35
-
0.13
-
60
-
-
3
-
0.21
3
3
4
5.8
3
I
6.0
0.07
4
FINDINGS
52.0
6.5
PATHOLOGICAL
0.18
VALUES,
52.0
LABORATORY
TABLE OF NON-TOXIC
“COLD”
N P
N
Solid and microfollicular adenoma Normal tissue ( ?) ~acrofollicular goitre Microfollicular goitre Follicular adenoma Microfollicular goitre Microfollicular adenoma Microfollicular goitre Solid and microfollicular adenoma Microfollicular goitre -
I I CASES
N P N I? N P N P N P N P
IN
Microfollicular adenoma Microfollicular goitre Solid and microfollicular adenoma Colloid goitre Solid and microfollicular adenoma Normal tissue Solid and microfollicular adenoma MicrofolIicular and colloid goitre Microfollicular adenoma Microfollicular and colloid goitre
RESULTS
P N P N P N P N P
AND
31.3 23.2
IO
13 5' 38 24
36 24 54 28
70
25 -
27 19
6
30 27 27 23
14 23
MIT
2.5 36.5
8
31 20
13 38 21 6
55 16
30 39 20
34 33 32
21
zd;
54
42 54
DIT
12.2 _
II.0
77
9
13.2
18
2.5 31
-. -
0.72
0.20
.--
_-
7
-
T,
-
5
18
5
40 4 -
T,
in thvroid
NODULE
lzsI com4ounds
THYROID
-
-
2.8
2.9
7 18
-
II -
22
tissue,
-
-
5.9 7.2
24 -
-
8 29
41 27 7
-
I.96 0.84
I.2
I.2
3.50 0.65 I.50 4.10 0.8 0.6 8 1.2
6.83 0.00
0.59
1.20
0.17
1.10
0.44
I.10
0.55
0.33 0.43
MIT/D11 rafi0
of told
62.0
15
53
‘7
San
19
46
4’
44
16
18
49
/a
3
TABLF:
0’
46.0
‘4
111
61.j
r.3
Mean
68.0
uptake 24 h %
1311
12
.vo.
-
___-
0.44
0.11
0.18
0.05
0.23
0.06
-
--
7.4
-
7.5
5.3
9.0
4.4
0.08
0.96
7.0
Fp*
PBI
0.17
PB’“l/ 48 h %I1
5
L
14
z
2
---
.._ - .._.. _
Cold nodule Cold nodule Cold nodule Hot nodule Cold nodule
adm.
2
nodule Warm nodule
__.-.
3
I
2
Hot
Warm nodule Hot nodule
a&z.
Da)ls after lZSI
______.. -_
PhotoSCalZ
._
P
N P N P N P N P N P
N P
N P N P N I? N P
_~_
__. findings
~_._
._..-
adenoma
Normal tissue Microfollicular
6 41
36 .j8.8
55
45 _~
-
38
61 -
45
44 __
42 -
cyst
Normal tissue Haemorrhagic
_
40.5 34.2
58 44 23 20 35 40 45
DIT
~___ ...___
IS
19
23 16 17 25 jz,
MIT
27 -
Normal tissue Embryonal adenoma Normal tissue
~__
.-___
---_
9.6
43
-
-
.-
---
5 _
to.9
11.7
9 -7 31 ---31 L.
T,
x.4
-
~-
-
-
7 _-
-
-
-~-
T,
--
---
-
-
_
G.6
2.5
-..2 10 -__
U, 2
6 I.84
-
0.9
0.44 I.1
MITIDIT ratio
0.8
-
2
~-
--
c
0.5’ 0.60
1.25 0.00 0.30 0.70
0.51
0.36
0.40
MI T/DIT ratio
oftotal
- .,
4.7 ‘4
6 17 36 -~ _
-
U,
lzaI compounds in thyroid tissue, “d
Solid and lnicrofollicul~r Normal tissue Microfollicular adenoma
atlenoma
--___
Microfollicufar adenoma Mixed micro- and macrofollicular goitre Mixed micro- and macro- fol. adenoma Microfollicular goitre Mixed follicular adenoma Cofloid go&e Mixed follicular adenoma Colloid goitre
Pathological
.__---._
Tissue
._
NODULAR
GOITRE STUDIES
131
the nodular tissue, in spite of the fact that the specimens were examined on an average two days after lz51 administration, against 3 days in the case of the cold nodules. The two unknown compounds were again found in three cases.
a
Fig. I. Autoradiogrsm of thin-layer chromatogram in two dimensions on mixture of Silica gel G and DEAE cellulose. First solvent system n-butanol-acetic acid-water (78: 5: ~7, v/v). Second solvent system n-butanol saturated with 2 -4’ ammonia. Butanol extracts of hydrolysates of thyroid tissue a: case I N; b: case4 P of Table I. CEin. Chim. A&,
20 (1968) 127-134
132
LEVIS
et id.
Table III summarises the results in 5 cases where the paranodular tissue was normal, including case 3 from the cold nodule group. The MIT/DIT ratio was lower than 1.1 in all except the last case, in which the high value found is probably accounted for by the high conversion of DIT to T, and hence the relatively low DIT concentration, The two unknown compounds have not been detected. Representative chromatograms with and without the two unknown compounds are shown in Figs. ra and b. The methanol-ammonia solution of the butanol extract of the hydrolysates of the tissues which contained the unknown spots were extracted with chloroformmethanol as described under METHODS. In all cases the radioactivity of the unknown spots was extractable into chlorofo~. This is shown in Fig. z where both the butano1 extract and the chloroform extract are chromatographed on the same plate. In order to add more information on the nature of the unknown lipids these were subjected to mild alkaline and to acid hydrolysis. It was found that the mobility on thin-layer plates of the two unknown spots was unchanged following mild alkaline hydrolysis but after acid hydrolysis only one spot identical to the iodide was detected. The major radioactive spot of the chloroform extract, when chromatographed on silicagel-G, presented a mobility very similar to that of phosphatidylcholine.
Fig. 2. Autoradiogram of thin-layer chromatogram on mixture of Silica gel G and DEAE) cellulose. Solvent system n-butanol saturated with z N ammonia. A, B, C are butanol extracts of the hydrolysates of cases 14 N, 14 P of Table II and 4 P of Table I. A’, B’, C’ arc the respective chloroform extracts. DISCUSSION
The finding in cold nodules of decreased transfer of iodine from MIT to the more heavily iodinated DIT, and hence a high ~IT~DIT ratio, is not surprising,
IiODULAR
GOITRE
STUDIES
I33
having been previously reported tive stage of iodine metabolism
by others l”$ll. The formation and hence a high MIT/DIT
of MIT is a more primiratio may be found in
several thyroid disorderP : in cold nodules, as here, after goitrogen administration13p1*, in experimental iodine deficiency15v1B, and in spontaneous human iodine deficiency goitrel7. The more active the thyroid, as evident from the 1311 uptake, the more DIT and T, are formed’*. In endemic goitre the low DIT and low iodothyronine concentrations are probably due to the dilution of the thyroidal iodine in a greater tissue mass, since the total iodine content of the gland may be normal, but the iodine concentration per unit mass is reduced16. Furthermore, the studies of Ermans et ~1.~~suggest that this decreased transfer iodothyronines is secondary
of iodine from MIT to DIT and from iodotyrosines to to the goitrous process rather than a primary causative
factor. This is in agreement with the finding by Dimitriadou et aLzo in Thailand that in diffuse goitre of young persons the MIT/DIT ratio is normal, whereas in nodular goitres of older individuals the rate of iodothyronine formation is low, with high MIT/DIT and iodotyrosine/iodothyronine ratios. The finding of unknown iodinated compounds which could be separated by lipid solvents in certain tissues of the examined goitres seems to be interesting. These unknown compounds, besides being lipid soluble, when taken from different subjects, have a definite mobility on thin-layer chromatography, moving faster than T, in the solvent
butanol-ammonia, but not in the solvent front. The occurrence of iodinated lipids has been reported
only after in vitro incu-
bation of 1311 with thyroid cell-free particulate fractions. Thus, De Groot and Carvalhozl suggested the formation of an iodinated lipid compound under the above mentioned experimental conditions, which moved to the solvent front on chromatog, raphy with butanol-ethanol-ammonia. Suzuki et n1.22, under similar conditionsreported the formation of both nonphospholipids and phospholipids which were isolated with chloroform extraction. These compounds, when chromatographed in butanol-dioxaneeammonia, also moved near the solvent front. It must be stressed that inorganic oxidation The lipid-soluble iodinated compounds presented preparations
and specifically
not in the normal
of iodide may produce artifactsz3. here are not found in all thyroid ones, experimentally
treated
in the
same way. In addition, these compounds are found mainly in the paranodular tissue of cold nodule cases. These findings exclude the possibility that unknowns U, and U, are artifacts produced in vitro. The spot usually
found
in larger
quantities
(U,) when chromatographed
in
systems for lipid separation, displays a single spot with mobility similar to phosphatidylcholine. This finding together with the fact that this compound is resistant to mild alcali predicates that the iodide-binding compound is a polar lipid, wherefrom iodide can be removed by acid treatment either through abolishing the binding capacity or through acid hydrolysis of the lipid. Vilkkiz4 has reported that specifically thyroidal lecithin comprises a fraction binding in vitro iodide and this is the conclusion reached by Schneider and WolfP, who also presented evidence suggesting reversible binding of iodide through electrostatic forces. The iodinated lipid compounds described here do not seem to bind iodide reversibly since iodide is not removed during washing of the chloroform phase. It may, therefore, be suggested that the thyroids with the unknown compounds contain increased amounts of such a specific lipid fraction which could bind iodide C&z. Chim. Acta,
20 (1968) 127-134
mms
I34 irreversibly, thus resulting in withdrawal able for thyroid hormone synthesis.
of iodide which, otherwise,
et al.
would be avail-
It is not known whether the formation of iodinated lipids is of primary importance in the aetiology of non-toxic goitre or whether it is secondary to the goitrous process pev se, as suggested by Ermans et al. IQ for the high MIT/DIT ratio. At anp rate, occurring
in a functionally
deficient
gland it would aggravate
the difficulty
in
the biosynthesis of thyroid hormones, diverting iodine from the normal metabolic pathway. Further studies are required in order to elucidate the role of these iodinated compounds in the generation or maintenance of human goitre. ACKNOWLEDGEMEXTS
This work was been supported National
Institutes
of Health,
by Grants No. AM 07464 and AM 08987 from the
Bethesda,
Md., U.S.A.
REFERENCES I R. PITT-RIVERS AND J. E. RALL, Endocrinology, 65 (1961) 309. 2 E. J. WAYNE, D. A. KOUTRAS AND W. I). ALEXANDER, Clivlical Aspcts 0.1Iodine lWetaholism, Blackwell, Oxford, 1964. 3 J. E. RALL, J. ROBBINS AND C. G. LEWALLEN, in G. FI~cus, K. V. THIMANN AND E. B. ASTWOOD (Eds.), The Hormones, vol. V, Academic Press, New York and London, 1964, p. 159. 4 R. PITT-RIVERS, D. HUBBLE AND W. H. HOATHER, J. Clin. Endocrinol., 17 (1957) 1313. 5 A. M. ERMANS, J, E. DUMONT AND P. A. BASTENIE, J. Clin. Endocvinol., 23 (1963) 539. 6 B. K. MALAMOS, G. K. DAIKOS, V. SAMARA AND D. A. KOUTRAS, Acta Endocrinol., 32 (1959) 31, 7 B. K. MALAMOS, J. KIBURIS AND G. LEVIS, journr’es Hell&es de S&wation Imme’diat! et dr Chvomatogvaphie, (IIIt?me J.I.S.I.C.), Association of Greek Chemist, Athens, 1965, p. Irg. 8 J, FOLCH, M. LEES AND S. G. H. SLOANE, ,I. Viol. Chem., 226 (1957) 497. 9 C. MIRAS, J. MANTZOS AND G. LEVIS, Biochen?. J.. 98 (1966) 782. IO G. B. SALABE, S. TONELLI, H. SALABE AND L. BASCHIERI, in C. CASSANO AND M. ANDREOLI (Eds.), Czwvewt Topics in Thvroid Research. Academic Press, New York and London, 1965, p. 900. II H. W. WAHNER, E. GAITAN AND P. CORREA, /. Clin. I
Acta,
20 (1968)
127-134