326 Horm. Metab. Res. 9 (1977) 326-332
© Georg Thieme Verlag Stuttgart
Anti Thyroid Hormone Autoantibodies under Experimental and Clinical Conditions R. Hehrmann, B. Höffken, A. von zur Mühlen, H. Creutzig+, J. Thiele++ and R.-D. Hesch Section of Clinical Endocrinology, Department of Internal Medicina, Department of Nuclear Medicine+ and Institute of Pathology++, Medical School, Hannover, Germany
cmft, Scaramuzzi and Blake (1975) reported on a long tenn experiment with active immunization Autoantibodies against thyroid hormones were experimenagainst testosterone. Elevated LH eoncentrations octally induced and elinieally observed. Rats were immunized with a single dose of T 3-conjugate, T 4-conjugate or both. curred after booster injeetions, but were only tranSix months later total T 3 and total T4 were measured by sient. Biological neutralization of testosterone was radioimmunoassays after alcohol extraetion as weil as TSH also eoneluded to be transient; these studies, howin rats (rTHS). Also antibody titers against T 3 and T4 were determined. Oxygen eonsumption of immunized and control ever, do not supply eonsistent data on the biological rats was measured in special metabolie cages. After immuniza- effeets of the exeessively inereased honnone eoneention Tl and/or T4 eoncentrations were markedly elevated trations, which were elosely eorrelated to the antiin close corretation to the respective antibody titers. rTHS body titers (Thorneycraft et al. 1975). Nieschlag was not changed. Oxygen consumption was not altered in et al. (1973) could not deteet signs of hyperthyroidimmunized animals, whereas thiamazol treated rats showed ism in the immunized rabbits as judged from weight derninished oxygen consumption indicating deereased basal metabolie rate. Clinically oceurring thyroid hormone autoand general appearanee. This publieation presents antibodies can be deteeted by RIA using charcoal separation data on total T 3, T4 and rTSH eoneentrations and for bound and free hormone since they result in high unspeT 3 and T 4 antibody titers in rats several months cifie binding.. Such sera äre subjeeted to antibody titer deafter a single immunization with protein conjugates termination and"total T 3 and T4 are measured after aleohol extraction. T 3 autoantibodies were observed in euthyroid of thyroid honnones. and hyperthyroid sera, and their titers were again correiated to total T 3 concentrations. It can be eoncluded, that the increased total hormone concentrations in antibody positive sera are biologically irrelevant, since rTSH and oxygen eonsumption were normal suggesting normal free hormone concentrations. In patients with thyroid hormone autoantibodies the diagnosis cannot be made from total hormone concentrations or antibody titers, but must be made on the basis of clinical appearanee and TRH tests.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Summary
In eontrast to most findings published so far we spent particular eare on the evaluation of the biological effeet of the raised honnone eoneentrations by measuring oxygen eonsumption of rats in a specially eonstrueted metabolie eage (Holtkamp, Ochs, Pfeiffer and Heming 1955, Harvey 1958, Denckla 1970). Under appropriate eonditions minimal oxygen eonsumption is an index of thyroid status (Denckla and Key-Words: Thyroid Hormone Autoantibodies - Total T 3 Marcum 1973) similar to the basal metabolie rate Total T4 - Active Immunization - Oxygen Consumption in man (Fraser and Nordin 1955). Staeheli, Val/oton and Burger (1975) reported the deteeIntroduction tion of thyroid honnone autoantibodies in patients with different thyroid functional status, Le. in Graves' Honnone autoantibodies have been experimentally disease, in multinodular euthyroid goiter, in primary indueed in animals after aetive immunization. Niehypothyroidism and in a ease of seeondary hypothy. schlag, Hemnann, Usadel, Schwedes, Schö[fling and roidism treated with dissieated thyroid. Their elinieal Kriiskemper (1973) described inereased total T 3 and study did not suggest a elose correlation between total T4 concentrations in rabbits 7 weeks after aetive thyroid honnone levels and elinical appearanee in paimmunization with thyroid honnone eonjugates and tients with antibody positive sera. histometric signs of thyroid gland hyperaetivity. The clinieal part of our study is being presented to Analogous results eould be demonstrated by the demonstrate, that with T 3 and T4 radioimmunoassame group (Nieschlag, Usadel, Kley, Schwedes, say (applying 8-anilino-naphthalene-sulfonie acid Schöffling and Kriiskemper 1974) 7-14 weeks after (ANS) to block TBG binding and dextrane eoated immunization with steroids. In their study an inereased LH- and FSH-seeretion foUowing testosterone ehareoal to separate bound and free honnone (Mitimmunization was also found. Thorneycraft, Thorney- suma, Colucci, Shenkman and Hollander 1972) thyroid honnone antibodies ean easily be deteeted by produeing high unspecifie binding (UB). Sera Supported by the Deutsche Forsehungsgemeinschaft, Bad with signifieantly elevated UB in our laboratory are Godesberg therefore submitted to antibody titer detenniantion and to aleohol extraetion with subsequent measureReceived: 5 May 1976 Accepted: IDee. 1976 ment of total T 3 and T4.
Anti Thyroid Hormone Autoantibodies under Experimental and Clinical Conditions
327
Materials and Methods Animal experiments, assay protocol: Four groups (A-D) of female albino Wistar rats (n = 6-8) weighing 230-270 g at the beginning of the experiment were housed in our own light and temperature controlled animal rooms (light from 7-19 h). They were immunized once by intracutaneous multiple si te injection of the back. 6 months later minimal oxygen consumption was measured and the rats were then bled at the same time to determine total T 3, total T 4, rTSH and T 3 and T 4 antibody titers.
T 3 methyl ester-BSA conjugate and T 4 eth~ster-BSA conjugate were used as specific antigens. The T3-ester contained less than 0.1% of T 4 and the T 4 ester less than 0.1% of T 3. After carbodiimid coupling to BSA and purification by dialysis, conjugates were estimated to contain 1 Mol T 3/T4 per 2 Mol of BSA. * Group A was injected with 1 mg of T 3 conjugate in 0.2 ml of 0.9% NaCI suspended with 0.2 ml of complete Freund's adjuvant. Group B received 1 mg of the T 4 conjugate, group C both T 3 and T 4 conjugates in the same volumes of saline and adjuvant. Group D served as controls and was injected with solvent and adjuvant alone.
Oxygen consumption: Metabolie cages were constructed as a modification of the methods of Harvey (1958) and Denckla (1970). They consisted of a cylinder, total volume being 600 ml and were connected to a manometer as a closed system. CO 2 was adsorbed by soda lime (Merck, Article No 6839), so that the difference between pressures before and after an interval of 10 min represents oxygen consumption (Fig. 1) .. The temperature was kept constant by a surrounding water bath at 3l 0 e. To minimize motor activity of the rats during the measurements, the surroundings was carefu11y kept quiet and the animal were observed, so that irritated and moving rats could be excluded or the measurements be repeated. Oxygen consumption in ml 02 /kg/h was calculated according to the fo11owing formula (Holtkamp et al. 1955): (VolCh - bw) x 273 x h x 6 2500 x T x bw Volch bw h T
Volume of the cylindric chamber (600 ml) body weight difference of manometer readings before and after oxygen consumption total temperature (31 0 C = 304 0 absolute)
T 3 and T 4 determination: The routine radioimmunoassay for total T 3 and T 4 used in our laboratory has been described in detail (Hehrmann and Schneider 1974). For determinations in autoantibody containing sera this method has been modified by an antecedent alcohol extraction with 1: 2 v/v 95% ethanol. No ANS is applied, when serum extracts are being determined. 100 fJl of extract for the T 3 assay and 20 fJl for the T 4 assay are added directly to the assay. Standard curves are run in same volumes of alcohol extracted T 3/T 4 free rat serum, since unspecific binding, especially of 12SJ-T4 , is different from buffer medium. All sampes are assayed in duplicate; bound and free hormone are separated by dextrane coated charcoal.
T 3 and T4 antibody determination : Without pretreatment a11 sera were diluted to final dilutions *T 3/T4 conjugate and specifications were kindly supplied by Henning Berlin.
•
water bath 31
soda
oe
fime
Fig. 1. Schematic drawing of the metabolie cages to mellsure oxygen consumption consisting of a cylindric chamber with sodium lime at the bottom to absorb CO 2 . The chamber is connected to a manometer as a closed system and is surrounded by a water bath to keep temperature constant. Oxygen consumption can be read from the manometer and is then calculated as ml O 2 per kg body weight per unit of time. of 1: 100, 1:500 and I: 2500 in the routine assay system (barbital buffer, pH 8.4) with 350 pg ANS per incubation tube in the case of the T 3 assay and 70 pg per tube in the T 4 assay, the incubation volume being 500 fJl. Separation was performed by dextrane coated charcoal. Titers - shown in Table 1 - are defined as that dilution of antiserum, which bound 50% of labe11ed T 3 or T 4.
rTSH determination: rTSH was determined by radioimmunoassay using the instructions and reagents (anti rTSH serum, rTSH for radioiodination, and rTSH standard) kindly supplied by the NIAMD. Histological examinations: Light microscopy of serial sections of the thyroid glands of all animals was performed using various staining methods (Haematoxylin Eosin, van Gieson, periodic acid Schiff reagent (PAS), Silver impregnation after Gomori and Movat). Clinical studies: In o ur routim radioimmunoassay for T 3 and T 4 antibody positive sera were simply detected by their unspecific binding, which is significantly higher than the assay VB, measured in T 3/T 4 free normal serum. Sera from patients with high VB are alcohol extracted as described above. Total T 3 and T 4 level are measured in the extracts, and serum dilutions are prepared to determine antibody titers as described for the rat sera. During the last 4 months, 5 antibody positive sera have been 0 bserved in our laboratory. The diagnosis of these patients were based on history , clinical signs, radioiodine thyroidal uptake and TRH test.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Immunization:
328
R. Hehrmann et aL
! i!SO 50
l1
I
1 I
1
II1II
J.. 1 " .,'
13 • normal
range
I
I
1$
15
10
r"
* ,,},
T,,
immuniz~
"3 .7i~
1"-.
thiamazol lI,d 32d
50
Fig. 2. Oxygen consumption of rats under resting conditions in control rats, T 3 -immunized, T4 -immunized and T 3 + T 4 immunized animals. Tbe two barrs at right represent oxygen consumption of thiarnazole treated animals.
25
Antithyroglobulin antibodies were also determined (Tbyroglobulin Haemagglutination Test Kit, WeUcome). ~-
Results The weight of all rats increased without differences between the groups to 360-470 g per animal at the end of the 6 months period. Three rats died du ring the experiment (No. 3, 18 and 29); all surviving animals were in good condition. Oxygen consumption: Oxygen consumption of control rats was 42.4 ± 6.1 ml/h/kg body weight. As shown in Figure 2, there was no significant difference between immunized rats and controls. In contrast, we measured significantly lower values in another group of thiamazoie treated (0.02% solution as drinking water ad lib. for 14 and 32 days resp.) aninlals: after 14 days oxygen consumption was 31.1 ± 5.0 ml 02/h/kg and after 32 days 28.0 ± 5.6 ml 02/h/kg. Histological findings: Light microscopical examinations of the thyroid glands of all anintals did not reveal any signs of increased or markedly reduced endocrine activity.
~imrrNJrIized
Fig. 3. Total T 3 and total T4 concentrations in T 3-, T 4and T 3 + T 4 immunized rats. Tbe shaded areas indicate the range of T rand T 4 -concentrations oe the normal control group.
not reached (Table 1). rTSH concentrations were not significantly altered as compared to the control group. Changes after immunization with T 4 : Immunization with T 4 conjugate alone caused a marked elevation (p < 0.005) of total T4 by approximately 50 ng/ml (Table 1, Fig. 3). Again this was correlated to the anti T 4 titers (r = 0.92, P < 0.001); all animals had anti T4 titers between 1:350 and 1:1200. T 3 increased slightly, but significantly (p < 0.01) and all sera showed increased binding of labelled T 3 without reaching 50%. rTSH concentrations were neither changed in this group. Changes after immunization with T 3 and T4 :
All animals had markedly eie va ted total T 3 (p < 0.005) concentrations and more slightly increased Changes after immunization with T 3 : T 4 concentrations (p < 0.05). Anti T 3 titers were Table 1 and Figure 3 indicate the almost 40-fold in- between 1: 290 and 1: 11 00 and anti T 4 titers becrease (by + 23 ng/ml) of total T 3 (p < 0.001) after tween 1: 120 and 1: 1000. Hormone concentrations active immunization with T 3 conjugate alone, which and antibody titers were less closely correlated than was accompanied by a slight but significant (p < after immunization with only one antigen (T 3: r = 0.01) increase of total T4. The rise of total T 3 was 0.79; P < 0.025. T 4: r = 0.73; P = 0.05). rTSH correlated to the anti T 3 -titers (r = 0.80, P < 0.025), concentrations were not different from controls. which were detectable in all animals between 1:350 and 1: 2500. All but 1 serum showed slightly increased Clinical results: binding of labelled T4 , although a 50% binding was During the last 4 months appr. 2400 sera were
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Controls T3 -
Anti Thyroid Hormone Autoantibodies under Experimental and Clinical Conditions
329
Table 1. Total T 3, total T 4 and rTSH concentrations oe groups A-D oe rats. Group A was immunized with T 3-conjugate, group B with T 4 conjugate alone, group C with both Tl" and T4-conjugates and group D were controls. Column 4 and 5 show anti-T 3/T4 titers (that is the serum dilution, at which 50% oe labelled T3/T4 were bound) or they show (in the case of those sera, where antibody concentrations were not high enough to determine titers) the percent binding of labelled T 3(f4 at 1:100 dilution. T 3 (ng/ml)
1
27.0 30.0 30.0 11.0 28.2 21.0
2 4 5
A
6 7
x 24.53 SD 7.41 s.e.m. 3.03
A
8 9 10 11 12 13 14
X 1.26 SD 0.35 s.e.m. ± 0.13
15 16 17 19 20 21
X 10".25 SD ± 4.04 s.e.m. ± 1.65
C
22 23 24 25 26 27
±
D
30
X
0.68 ±0.25 s.e.rn. ± 0.05
SD
54.3 17.3 7.05
± ±
89.4 45.0 17.0
±
56.45 25.2 20.3
34.6 2.1 0.75
± ±
1: 1375 821 335 15+ 8
298 370 310 300 134 204 435
±
±
293.0 99.7 37.7
7
6 4 25 13
± ±
236 184 75
147 328 295 391
± ±
253 94.6 33.4
examined for T 3 5 antibody positive sera were detected, 2 of which were from a patient before and after therapy (case 1, see below). The unspecific binding of these sera was markedly increased (3073%). Normal sera and all other sera from patients with euthyroid goiter, hyper- and hypothyroidism bad unspecific bindings between -2% and +4% of the assay VB, which is determined in T 3fT 4 free serum.
±
±
1150 650 900 550 600 1200 700
1:
821 266 100
480 700 290 400 1100 650
1: 1: 1: 1: 1: 1:
1:
603 287 117
± ±
±
165 700 180 120 420 1000
1: 431 354 144
1+
o
2
1 2.5 3 2.5 1
1.5 3 2.5 2 1 1.5
282
1: 1: 1: 1: 1: 1: 1:
1: 1: I: 1: 1: 1:
± ±
140 290 153
±
t
±
9.6 7.3 2.96
±
11.1 7.2 2.7
±
425 110 373 50 50 412
35.4 34.8 36.0 32.4 31.5 32.7 36.6 37.2
± ±
50.9
6.5+ 14.5 3.5 14.5 0.5 18.5
1: 800 1: 1700 1: 2500 1: 350 1: 2000 1: 900
239.7
± 124.7
55.5 51.0 40.5 43.8 41.4 106.5
± ±
anti-T3-titer anti-T4-titer or +%B 1:100 or ~B at 1:100
395 180 130 400 182 145
150.0 63.3 63.3 60.0 60.0 160.0 69.3
0.69 0.60 0.63 0.45 0.54 0.87 0.78 0.87
28
D
±
11.40 11.40 3.84 11.7 15.6 7.56
C
rTSH (ng/ml)
44.4 87.9 50.4 50.4 53.7 39.0
1.65 1.26 0.99 1.17 0.66 1.56 1.50
B
B
T 4 (ng/ml)
1.75 0.75 0.21
o 3
1.63
± 1.27 ± 0.45
Case 1 was a 53 year old woman, whose serum was examined he fore and 6 months after radioiodine treatment for clinical hyperthyroidism, which was proven by negative TRH tests and accelerated intrathyroidal radioiodine exchange. After alcohol extraction total T 4 was 225 ngJml and T 3 was 6.7 ngJml before treatment. Without alcohol extraction, T 3 was below the limit of detection (0.2 ng/ml), when correction was made for the assay UB instead for the high VB (45%) of this individual serum.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
No
R. Hehrmann et aL
6 months after treatment the patient was clinically improved although still hyperthyroid. Total T 4 after extraction was now 55 ng/mI and T 3 was 3.3 ng/mI. The T 3 antibody dilution curves of this serum before and 6 months after radioiodine treatment are shown in Figure 4 as compared to an antibody negative con· trol serum. Binding of labelIed T 4 (4%) was not in· creased before and after treatment.
1:
.-
25
50
100
250
500
dilution of "serum
Fil. 4. T 3 antibody determination of case I, demonstrated as % binding of 125J-T3 by different dilutions of patients's serum. o before treatment with radioiodine, clinically hyperthyroid, total T 3 = 6.6 na/ml ~ 6 months after treatment with radioiodine, clinica1ly sUghtly hypothyroid, total T 3 = 3.3 na/mt + Antibody negative controt, normal serum, total T 3 =
1.1 ng/mL
Case 2 is a 40 year old woman with no clinical signs of thyroid dysfunction. T 3 after extraction was 0.7 ng/mI, T 4 was 67 ng/mI, both values being in the lower normal range. Unspecific binding for T 3 was 30.5% at 1: 10 and for T 4 4% at 1: SO dilution. The TSH concentrations of this patient was borderline, but showed exaggerated response to TRH (TSH o = 6.6, TSH 30 = 32 #lU/mI). Case 3 is a 28 year old patient 5 years after treatment with thiamazoie and thyroxine for hyperthy· roidism. She was now clinically euthyroid without treatment. Total T 3 was 5.03 ng/ml, total T 4 was 87 ng/mI after extraetion. Inereased binding of labelIed T 4 could not .be demonstrated, however, 32.7% of labelIed T 3 were bound at 1: 10 dilution She had anormal TSH response to TRH (TSHo = 5.2, TSH 30 = 12.9 #lU/mI). Case 4 is a 30 year old woman with mild elinieal signs of hyperthyroidism. Total T 3 was 23.5 ng/mI and total T 4 was 88 ng/mI after extraetion. Binding of labeUed T 3 was 73.5% at 1: 10 dilution and of T. was 4% at 1: 50 (this patient did not attend our clinic, a TRH test was not performed).
It should be noted, that all of these patients did have variable eoneentrations of anti T 3 autoantibodies, but no T 4 antibodies. Also they had no an· tithyroglobulin antibodies (TRC-test positive between 1:1 and 1:5). Antimicrosomal antibodies have not been determined.
Discussion Previous studies of Nieschillg et al. (1973), Herrmann, Rusche and Krüskemper (1975) and many others, who investigated animals at relatively short intervals after active immunization with hormones suggested neutralization of endogenous hormones by the antibodies produeed, subsequent stimulation by an increase of pituitary hormones leading to markedly elevated levels of the peripheral hormone. Despite the high total hormone concentiations there were °no clinieal signs of hyperfunetion and despite the high antibody titers, there were no signs of hypofunction except for the mild elevation of LH after testosterone immunization. However, Thomeycrajt et al. 1975 demonstrated in a long term study, that LH changes were only transient. This study coneentrated on a late time interval after imrnunization with thyroid hormones and on the quantitation of biological hormone action. For this purpose the determination of oxygen consumption of immunized rats as a parameter of the basal meta· bolie rate and as an index of the thyroid status was chosen using the metabolic eages described. It can clearly be stated, that 6 months after active immunization with T 3 and/or T 4 all animals had cir· culating autoantibodies of measurable titer and in· creased total hormone eoneentrations, whieh were partially excessively high. It may seem to be surprising that the T 3 immunized animals had also slightly inereased T 4 eoneentrations and increased binding of labelIed T 4 indicating small amounts of T 4 antibodies. The T 4 immunized animals had also elevated T 3 levels and T 3 antibodies of low eoncentrations. One explanation could be, that the T 3 conjugates were not free from T 4 contamination and the T 4 conjugates from T 3. However, the rabbits, immunized with the same material had highly speeifie antibodies now used in our RIAs (Hesch and Hüfner 1972, Hehrmllnn and Schneider 1974). It seems more likely, that the rats did not produee highly speeific antibodies. The results ean best be explained by a crossreaction of the 10w affmity T 3 antibodies with T4 and viee versa of about 1-3%. Despite the pre· senee of thyroid hormone autoantibodies and high total hormone eoncentrations, all immunized animals had the same weight development and the same oxygen eonsumption as the eontrols with normal hor· mone eoneentrations and no antibodies. Also they had unchanged rTSH levels as an indicator for thyroid function.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
330
In connection with the various studies of other groups already quoted one can assume the following time course of hormone and antibody production: In a first phase after active immunization antibody concentrations rise and may biologicalty neutralize endogenous hormone with transient decrease of free hormone concentrations. Assuming an intact feedback mechanism this leads to a stimulation of pituitary tropic hormone secretion and thereby to a stimulation of the peripheral gland, exhtbiting histometrical signs of hyperfunction (Nieschlag et al. 1973). Total hormone concentrations rise until alt of the antibody produced is saturated by hormone. This is particularly supported by the fmding, that total hormone concetrations are closely correlated to the antibody titers. At that time TSH or LH concentrations are no longer elevated. Booster injections again lead to antibody production, LH stimulation, further increase of testosterone levels etc. (Thomeycraft et al. 1975). At the time of our investigation the biologicalty ineffeetive secretion of hormone excess by the thyroid after immunization had already stopped, since antibody titers had not been raised by booster injections. A new equilibrium at a higher level of total hormone eoncentration was reached, the animals seemed euthyroid from weight development and general appearance, from oxygen consumption and rTSH coneentrations in eorrelation to histological fmdings. It must be assumed, that free hormone coneentrations, which have not been directly measured in this study, are normal at that time, since neither the central receptor shows any measurable signs of disturbance (normal rTSH) nor does the peripheral receptor (normal oxygen consumption). From these experimental results it can be coneluded that the degree of hormone elevation in antibody positive sera of patients does not give any information on the clinical status of thyroid function in these individuals. It does, therefore, not surprise, that thyroid hormone autoantibodies in connection with high total hormone eoneentrations (if measured after alcohol extraction) can be found in euthyroid, hypothyroid and hyperthyroid patients (Staeheli et al. 1975).
331
Radioimmunological determinations of T 3 and T4 in antibody containing sera are only reliable after alcohol extraction, since without extraction, the individual VB of that serum will be mueh higher than the assay VB and the results obtained will be falsely low or even not measurable. However, the diagnosis cannot be based on the - correet - total hormone eoncentrations either, since the greater part is neutralized by the cireulating antibodies. In fact 2 from our 4 patients were clinicalty euthyroid and case 4 with T 3 excess (23.5 ng/ml) was only slightly hyperthyroid. It is interesting, that case 2 with normal total T3 and T4 and low titer anti-T 3 -antibodies did have a pathological TRH test, indicating, that this patient may become clinically hypothyroid. It seems important to us that we could not detect T4 autoantibodies in human beings. Theoretically one could expect thyroid hormone aotoantibodies being the result of release of thyroglobulin from a diseased gland. This should, if at all, lead to antithyroglobulin and anti T4 antibodies, both of which were not deteetable. T 3 autoantibodies may, therefore, be of other origin than from the hapten thyroglobulin-T3. Conjugation of T 3 to peripheral immunoglobulins under pathological circumstances must be seriously considered as one possibility. The diagnosis of patients with T 3 and/or T4 antibodies must be made on the basis of the clinical fmdings and of the TRH test. From a clinical point of view the existence of thyroid hormone aotoantibodies does not seem to be rare. Since they can only be deteeted, if the individual unspeeific binding of each serum is determined, this should be routinely done for alt sera, that enter a T 3 /T 4 RIA laboratory. Several years ago we have already commented on the importance to determine V.B., because deviation from the blank binding was rather frequent (Hesch, Hüfner, von zur Mühlen and Köbberling 1974). However, these additional determinations would mean an extremely increasing expenditure, so that we claim for these additional investigations at least in all cases, where there are discrepaneies between clinical appearance, T 3 /T 4 concentrations and the results of the TRH test.
Referenees Denckla, W.D.: Minimal oxygen eonsumption in the female rat, same new definitions and measurements. J.AppLPhysiology 29: 2(;3-273 (1970) Denckla, W.D., E. Marcum: Minimal O 2 consumption as an index of thyroid status: Standardization of method. Endocrinology 93: 61-73 (1973) Fraser, R., B.E.C Nordin: The basal metabolie rate during sleep. Laneet I: 532-533 (1955) Harvey, D.G.: The measurement of oxygen eonsumption in small animals. J.Pharmacy Pharmaeology 10: 483-492 (1958) Hehrmann, R., C. Schneider: Der Radioimmunoassay fUr Trijodthyronin und Thyroxin im Serum und seine An-
wendung bei Hyperthyreosen. Radiologe 14: 156-160 (1974) Herrmann, J., H.J. Rusche, M. Berger, H.L. KTÜskemper: Thyroid funetion and triiodothyronine and thyroxine kineties in rabbits immunized with thyroid hormone. Aeta Endocrinol. (Kbh.) 78: 276 (1975) Hesch, R.·D., M. Hüfner: Highly speeific antibodies to triiOdothyronine. Acta BioLMed.Germ. 28: 861-867 (1972) Hesch, R.-D., M. Hüfner, A. von zur Mühlen, J. Köbberling: Radioimmunochemical measurement of thyroid hormones and thyroxine-binding globulin. IAEA-SM- 177/30 (1974) 161
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Anti Thyroid Hormone Autoantibodies under Experimental and Clinica1 Conditions
332
Sigbritt Werner, H. Löw
Holtkamp, D.E., S. Ochs, C. C. Pfeiffer, A.E. Heming: Determination of the oxygen consumption of groups of rats. Endocrinology 56: 93-104 (1955) MitaumJl, T. J. Colucci, L. ShenkmJln, C.S. Hollonder: Rapid simultaneous radioimmunoassay for trüodothyronine and thyroxine in unextraeted serum. Bioehern. Biophys.Res.Comm. 46: 2107-2114 (1972) Nieschlog, E., J. HerrmJlnn, X.H. Usadei, U. Schwedes, K. Schöffling, H.L. Krüskemper: Thyroid hypertrophy and hyperfunetion caused by aetive immunization with thyroid hormones. J.Endocr. 57: 555-556 (1973)
Horm. Metab. Res. 9 (1977) 332-336
© Georg Thieme Verlag Stuttgart
Urinary Excretion of Cyclic AMP and Cyclic GMP in Primary Hyperparathyroidism with Reference to Clinical Signs and Symptoms Sigbritt vyerner and H. Löw Department of Endocrinology and Metabolism, Karolinska Hospital, Stockholm, Sweden Summary 3S patients with surgica1ly proven primary hyperparathyroidims (PHPT) were evaluated with regard to urinary cyclie AMP and cyelie GMP excretion. They showed no signs of bone disease or impaired renal function. Preoperatively, cyelie AMP was increased in 28 patients (80%). Mean excretory value for the whole group was 8.5 ± 0.8 micromoles/g creat/24 hours, compared with controls, 4.2 ± 0.13 micromoles/g creat/24 hours. Cyclic AMP excretion was normalized after surgery.
Urinary cyclic GMP was increased in 14 patients (40%) compared with excretion rates of controls, 0.46 ± 0.03 micromoles/g ereat/24 hours. Cyclie GMP increments persisted postoperatively, although at a lower level. There was an apparent lack of effect of one nucleotide on the other. Clinical, pathological' and biochemical data were analysed and correlated for signifieant assoeiations. The patients demonstrating augmented cyclic GMP excretion rates were not distinguishable from those with normal cyclic GMP production rates with regard to S-calcium, U-calcium, Ucyclie AMP, S-phosphate, cholelithiasis, nephrolithiasis, hypertension or age. One of twelve patients meeting the criteria of normacalcemie PHPT had increased excretion of eyclie AMP and was submitted to parathyroid surgery. Microscopy showed four normal glands. Dur data indicate that the increased excretion of cyelic GMP in urine was not a metabolie efract of the parathyroid hormone, since increments were noted also after reseetion of the parathyroids. Furthermore, it is not likely that the cyelic GMP increments reflected an increased secretion of calcitonin, since 'these increments were noted during hypercalcemia as weil as normocalcemia. The source and cause of produetion of the cyelic GMP remain to be determined. Received: 30 June 1976
Accepted: 1 Dec, 1976
Key-Words: PrimJlry Hyperparathyroidism - Cyclic AMPCyclic GMP
Introduction Patients with primary hyperparathyroidism (PHPT) and nonnal renal function show similar disturbances with regard to calcium, phosphate parameters and parathyroid pathology irregardless of the presence or absence of a long history of repeated renal colics (Lloyd 1968, Wem er, Hjem and Sjöberg 1974). This fmding is of clinical importance, since "asym. ptomatic" PHPT will oe diagnosed in a steadily increasing number due to the use of serum multichannel auto-analyzers. It emphasizes also the fact that disclosing prevalent hypercalcemia will not help in the early diagnosis of PHPT. Patients with PHPT have usually increased excretion of cyclic 3'S'-adenosine monophosphate~yclic AMP) illustrating parathyroid honnone induced augmented adenylate cyclase activity in the kidney (Chose and Aurbach 1967, Kaminsky, Broadus, Hardman, Jones, Ball, Sutherland and Liddie 970, Melson, Chose and Aurbach 1970, Gill and Casper 1971). The analysis of urinary cyclic AMP can be a helpful diagnostic tool to exclude other hypercalcemic states (Kaminsky et al. 1970, Neelon, Drezner, Buch and Lebovitz 1973, Murad and Pak 1972). However, data are sparse showing the value of the cyclic AMP analysis in conflITIling the diagnosis of PHPT in patients with nonnocalcemia and active stone disease (Murad and Pak 1972, Schmidt-Gayk and Röher 1972).
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Nieschlag, E., K.H. Usadei, H.H. Kley, U. Schwedes, K. Schöf/ling, H.L. Krüskemper: A new approach for investigating hypothalamo-pituitary-gonadal and adrenal feedback control mechanism: Active immunization with steroids. Acta Endocrinol. 76: 556-569 (1974) Staeheli, v., M.B. Valloton, A. Burger: Deteetion of human anti-thyroxine and anti-triiodothyronine antibodies in different thyroid conditions. J .Clin.Endocrinol.Metab. 41: 669-675 (1975) Thorneycraft, IH., N.K. Thorneycraft, R.J. Scaramuzzi, ChA. Bloke: Radioimmunoassay of serum LH and testosterone in male rabbits aetively immunized against testosterone. Endocrinology 97: 301-306 (1975) Request for reprints should be adressed to: Dr. R. Hehrmann, Seetion of Clinical Endocrinology, Department of Internal Medicine, Medical School. Hannover, 0.3 Hannover (Germany)
© Georg Thieme Verlag Stuttgart
Anti Thyroid Hormone Autoantibodies under Experimental and Clinical Conditions R. Hehrmann, B. Höffken, A. von zur Mühlen, H. Creutzig+, J. Thiele++ and R.-D. Hesch Section of Clinical Endocrinology, Department of Internal Medicina, Department of Nuclear Medicine+ and Institute of Pathology++, Medical School, Hannover, Germany
cmft, Scaramuzzi and Blake (1975) reported on a long tenn experiment with active immunization Autoantibodies against thyroid hormones were experimenagainst testosterone. Elevated LH eoncentrations octally induced and elinieally observed. Rats were immunized with a single dose of T 3-conjugate, T 4-conjugate or both. curred after booster injeetions, but were only tranSix months later total T 3 and total T4 were measured by sient. Biological neutralization of testosterone was radioimmunoassays after alcohol extraetion as weil as TSH also eoneluded to be transient; these studies, howin rats (rTHS). Also antibody titers against T 3 and T4 were determined. Oxygen eonsumption of immunized and control ever, do not supply eonsistent data on the biological rats was measured in special metabolie cages. After immuniza- effeets of the exeessively inereased honnone eoneention Tl and/or T4 eoncentrations were markedly elevated trations, which were elosely eorrelated to the antiin close corretation to the respective antibody titers. rTHS body titers (Thorneycraft et al. 1975). Nieschlag was not changed. Oxygen consumption was not altered in et al. (1973) could not deteet signs of hyperthyroidimmunized animals, whereas thiamazol treated rats showed ism in the immunized rabbits as judged from weight derninished oxygen consumption indicating deereased basal metabolie rate. Clinically oceurring thyroid hormone autoand general appearanee. This publieation presents antibodies can be deteeted by RIA using charcoal separation data on total T 3, T4 and rTSH eoneentrations and for bound and free hormone since they result in high unspeT 3 and T 4 antibody titers in rats several months cifie binding.. Such sera äre subjeeted to antibody titer deafter a single immunization with protein conjugates termination and"total T 3 and T4 are measured after aleohol extraction. T 3 autoantibodies were observed in euthyroid of thyroid honnones. and hyperthyroid sera, and their titers were again correiated to total T 3 concentrations. It can be eoncluded, that the increased total hormone concentrations in antibody positive sera are biologically irrelevant, since rTSH and oxygen eonsumption were normal suggesting normal free hormone concentrations. In patients with thyroid hormone autoantibodies the diagnosis cannot be made from total hormone concentrations or antibody titers, but must be made on the basis of clinical appearanee and TRH tests.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Summary
In eontrast to most findings published so far we spent particular eare on the evaluation of the biological effeet of the raised honnone eoneentrations by measuring oxygen eonsumption of rats in a specially eonstrueted metabolie eage (Holtkamp, Ochs, Pfeiffer and Heming 1955, Harvey 1958, Denckla 1970). Under appropriate eonditions minimal oxygen eonsumption is an index of thyroid status (Denckla and Key-Words: Thyroid Hormone Autoantibodies - Total T 3 Marcum 1973) similar to the basal metabolie rate Total T4 - Active Immunization - Oxygen Consumption in man (Fraser and Nordin 1955). Staeheli, Val/oton and Burger (1975) reported the deteeIntroduction tion of thyroid honnone autoantibodies in patients with different thyroid functional status, Le. in Graves' Honnone autoantibodies have been experimentally disease, in multinodular euthyroid goiter, in primary indueed in animals after aetive immunization. Niehypothyroidism and in a ease of seeondary hypothy. schlag, Hemnann, Usadel, Schwedes, Schö[fling and roidism treated with dissieated thyroid. Their elinieal Kriiskemper (1973) described inereased total T 3 and study did not suggest a elose correlation between total T4 concentrations in rabbits 7 weeks after aetive thyroid honnone levels and elinical appearanee in paimmunization with thyroid honnone eonjugates and tients with antibody positive sera. histometric signs of thyroid gland hyperaetivity. The clinieal part of our study is being presented to Analogous results eould be demonstrated by the demonstrate, that with T 3 and T4 radioimmunoassame group (Nieschlag, Usadel, Kley, Schwedes, say (applying 8-anilino-naphthalene-sulfonie acid Schöffling and Kriiskemper 1974) 7-14 weeks after (ANS) to block TBG binding and dextrane eoated immunization with steroids. In their study an inereased LH- and FSH-seeretion foUowing testosterone ehareoal to separate bound and free honnone (Mitimmunization was also found. Thorneycraft, Thorney- suma, Colucci, Shenkman and Hollander 1972) thyroid honnone antibodies ean easily be deteeted by produeing high unspecifie binding (UB). Sera Supported by the Deutsche Forsehungsgemeinschaft, Bad with signifieantly elevated UB in our laboratory are Godesberg therefore submitted to antibody titer detenniantion and to aleohol extraetion with subsequent measureReceived: 5 May 1976 Accepted: IDee. 1976 ment of total T 3 and T4.
Anti Thyroid Hormone Autoantibodies under Experimental and Clinical Conditions
327
Materials and Methods Animal experiments, assay protocol: Four groups (A-D) of female albino Wistar rats (n = 6-8) weighing 230-270 g at the beginning of the experiment were housed in our own light and temperature controlled animal rooms (light from 7-19 h). They were immunized once by intracutaneous multiple si te injection of the back. 6 months later minimal oxygen consumption was measured and the rats were then bled at the same time to determine total T 3, total T 4, rTSH and T 3 and T 4 antibody titers.
T 3 methyl ester-BSA conjugate and T 4 eth~ster-BSA conjugate were used as specific antigens. The T3-ester contained less than 0.1% of T 4 and the T 4 ester less than 0.1% of T 3. After carbodiimid coupling to BSA and purification by dialysis, conjugates were estimated to contain 1 Mol T 3/T4 per 2 Mol of BSA. * Group A was injected with 1 mg of T 3 conjugate in 0.2 ml of 0.9% NaCI suspended with 0.2 ml of complete Freund's adjuvant. Group B received 1 mg of the T 4 conjugate, group C both T 3 and T 4 conjugates in the same volumes of saline and adjuvant. Group D served as controls and was injected with solvent and adjuvant alone.
Oxygen consumption: Metabolie cages were constructed as a modification of the methods of Harvey (1958) and Denckla (1970). They consisted of a cylinder, total volume being 600 ml and were connected to a manometer as a closed system. CO 2 was adsorbed by soda lime (Merck, Article No 6839), so that the difference between pressures before and after an interval of 10 min represents oxygen consumption (Fig. 1) .. The temperature was kept constant by a surrounding water bath at 3l 0 e. To minimize motor activity of the rats during the measurements, the surroundings was carefu11y kept quiet and the animal were observed, so that irritated and moving rats could be excluded or the measurements be repeated. Oxygen consumption in ml 02 /kg/h was calculated according to the fo11owing formula (Holtkamp et al. 1955): (VolCh - bw) x 273 x h x 6 2500 x T x bw Volch bw h T
Volume of the cylindric chamber (600 ml) body weight difference of manometer readings before and after oxygen consumption total temperature (31 0 C = 304 0 absolute)
T 3 and T 4 determination: The routine radioimmunoassay for total T 3 and T 4 used in our laboratory has been described in detail (Hehrmann and Schneider 1974). For determinations in autoantibody containing sera this method has been modified by an antecedent alcohol extraction with 1: 2 v/v 95% ethanol. No ANS is applied, when serum extracts are being determined. 100 fJl of extract for the T 3 assay and 20 fJl for the T 4 assay are added directly to the assay. Standard curves are run in same volumes of alcohol extracted T 3/T 4 free rat serum, since unspecific binding, especially of 12SJ-T4 , is different from buffer medium. All sampes are assayed in duplicate; bound and free hormone are separated by dextrane coated charcoal.
T 3 and T4 antibody determination : Without pretreatment a11 sera were diluted to final dilutions *T 3/T4 conjugate and specifications were kindly supplied by Henning Berlin.
•
water bath 31
soda
oe
fime
Fig. 1. Schematic drawing of the metabolie cages to mellsure oxygen consumption consisting of a cylindric chamber with sodium lime at the bottom to absorb CO 2 . The chamber is connected to a manometer as a closed system and is surrounded by a water bath to keep temperature constant. Oxygen consumption can be read from the manometer and is then calculated as ml O 2 per kg body weight per unit of time. of 1: 100, 1:500 and I: 2500 in the routine assay system (barbital buffer, pH 8.4) with 350 pg ANS per incubation tube in the case of the T 3 assay and 70 pg per tube in the T 4 assay, the incubation volume being 500 fJl. Separation was performed by dextrane coated charcoal. Titers - shown in Table 1 - are defined as that dilution of antiserum, which bound 50% of labe11ed T 3 or T 4.
rTSH determination: rTSH was determined by radioimmunoassay using the instructions and reagents (anti rTSH serum, rTSH for radioiodination, and rTSH standard) kindly supplied by the NIAMD. Histological examinations: Light microscopy of serial sections of the thyroid glands of all animals was performed using various staining methods (Haematoxylin Eosin, van Gieson, periodic acid Schiff reagent (PAS), Silver impregnation after Gomori and Movat). Clinical studies: In o ur routim radioimmunoassay for T 3 and T 4 antibody positive sera were simply detected by their unspecific binding, which is significantly higher than the assay VB, measured in T 3/T 4 free normal serum. Sera from patients with high VB are alcohol extracted as described above. Total T 3 and T 4 level are measured in the extracts, and serum dilutions are prepared to determine antibody titers as described for the rat sera. During the last 4 months, 5 antibody positive sera have been 0 bserved in our laboratory. The diagnosis of these patients were based on history , clinical signs, radioiodine thyroidal uptake and TRH test.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Immunization:
328
R. Hehrmann et aL
! i!SO 50
l1
I
1 I
1
II1II
J.. 1 " .,'
13 • normal
range
I
I
1$
15
10
r"
* ,,},
T,,
immuniz~
"3 .7i~
1"-.
thiamazol lI,d 32d
50
Fig. 2. Oxygen consumption of rats under resting conditions in control rats, T 3 -immunized, T4 -immunized and T 3 + T 4 immunized animals. Tbe two barrs at right represent oxygen consumption of thiarnazole treated animals.
25
Antithyroglobulin antibodies were also determined (Tbyroglobulin Haemagglutination Test Kit, WeUcome). ~-
Results The weight of all rats increased without differences between the groups to 360-470 g per animal at the end of the 6 months period. Three rats died du ring the experiment (No. 3, 18 and 29); all surviving animals were in good condition. Oxygen consumption: Oxygen consumption of control rats was 42.4 ± 6.1 ml/h/kg body weight. As shown in Figure 2, there was no significant difference between immunized rats and controls. In contrast, we measured significantly lower values in another group of thiamazoie treated (0.02% solution as drinking water ad lib. for 14 and 32 days resp.) aninlals: after 14 days oxygen consumption was 31.1 ± 5.0 ml 02/h/kg and after 32 days 28.0 ± 5.6 ml 02/h/kg. Histological findings: Light microscopical examinations of the thyroid glands of all anintals did not reveal any signs of increased or markedly reduced endocrine activity.
~imrrNJrIized
Fig. 3. Total T 3 and total T4 concentrations in T 3-, T 4and T 3 + T 4 immunized rats. Tbe shaded areas indicate the range of T rand T 4 -concentrations oe the normal control group.
not reached (Table 1). rTSH concentrations were not significantly altered as compared to the control group. Changes after immunization with T 4 : Immunization with T 4 conjugate alone caused a marked elevation (p < 0.005) of total T4 by approximately 50 ng/ml (Table 1, Fig. 3). Again this was correlated to the anti T 4 titers (r = 0.92, P < 0.001); all animals had anti T4 titers between 1:350 and 1:1200. T 3 increased slightly, but significantly (p < 0.01) and all sera showed increased binding of labelled T 3 without reaching 50%. rTSH concentrations were neither changed in this group. Changes after immunization with T 3 and T4 :
All animals had markedly eie va ted total T 3 (p < 0.005) concentrations and more slightly increased Changes after immunization with T 3 : T 4 concentrations (p < 0.05). Anti T 3 titers were Table 1 and Figure 3 indicate the almost 40-fold in- between 1: 290 and 1: 11 00 and anti T 4 titers becrease (by + 23 ng/ml) of total T 3 (p < 0.001) after tween 1: 120 and 1: 1000. Hormone concentrations active immunization with T 3 conjugate alone, which and antibody titers were less closely correlated than was accompanied by a slight but significant (p < after immunization with only one antigen (T 3: r = 0.01) increase of total T4. The rise of total T 3 was 0.79; P < 0.025. T 4: r = 0.73; P = 0.05). rTSH correlated to the anti T 3 -titers (r = 0.80, P < 0.025), concentrations were not different from controls. which were detectable in all animals between 1:350 and 1: 2500. All but 1 serum showed slightly increased Clinical results: binding of labelled T4 , although a 50% binding was During the last 4 months appr. 2400 sera were
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Controls T3 -
Anti Thyroid Hormone Autoantibodies under Experimental and Clinical Conditions
329
Table 1. Total T 3, total T 4 and rTSH concentrations oe groups A-D oe rats. Group A was immunized with T 3-conjugate, group B with T 4 conjugate alone, group C with both Tl" and T4-conjugates and group D were controls. Column 4 and 5 show anti-T 3/T4 titers (that is the serum dilution, at which 50% oe labelled T3/T4 were bound) or they show (in the case of those sera, where antibody concentrations were not high enough to determine titers) the percent binding of labelled T 3(f4 at 1:100 dilution. T 3 (ng/ml)
1
27.0 30.0 30.0 11.0 28.2 21.0
2 4 5
A
6 7
x 24.53 SD 7.41 s.e.m. 3.03
A
8 9 10 11 12 13 14
X 1.26 SD 0.35 s.e.m. ± 0.13
15 16 17 19 20 21
X 10".25 SD ± 4.04 s.e.m. ± 1.65
C
22 23 24 25 26 27
±
D
30
X
0.68 ±0.25 s.e.rn. ± 0.05
SD
54.3 17.3 7.05
± ±
89.4 45.0 17.0
±
56.45 25.2 20.3
34.6 2.1 0.75
± ±
1: 1375 821 335 15+ 8
298 370 310 300 134 204 435
±
±
293.0 99.7 37.7
7
6 4 25 13
± ±
236 184 75
147 328 295 391
± ±
253 94.6 33.4
examined for T 3 5 antibody positive sera were detected, 2 of which were from a patient before and after therapy (case 1, see below). The unspecific binding of these sera was markedly increased (3073%). Normal sera and all other sera from patients with euthyroid goiter, hyper- and hypothyroidism bad unspecific bindings between -2% and +4% of the assay VB, which is determined in T 3fT 4 free serum.
±
±
1150 650 900 550 600 1200 700
1:
821 266 100
480 700 290 400 1100 650
1: 1: 1: 1: 1: 1:
1:
603 287 117
± ±
±
165 700 180 120 420 1000
1: 431 354 144
1+
o
2
1 2.5 3 2.5 1
1.5 3 2.5 2 1 1.5
282
1: 1: 1: 1: 1: 1: 1:
1: 1: I: 1: 1: 1:
± ±
140 290 153
±
t
±
9.6 7.3 2.96
±
11.1 7.2 2.7
±
425 110 373 50 50 412
35.4 34.8 36.0 32.4 31.5 32.7 36.6 37.2
± ±
50.9
6.5+ 14.5 3.5 14.5 0.5 18.5
1: 800 1: 1700 1: 2500 1: 350 1: 2000 1: 900
239.7
± 124.7
55.5 51.0 40.5 43.8 41.4 106.5
± ±
anti-T3-titer anti-T4-titer or +%B 1:100 or ~B at 1:100
395 180 130 400 182 145
150.0 63.3 63.3 60.0 60.0 160.0 69.3
0.69 0.60 0.63 0.45 0.54 0.87 0.78 0.87
28
D
±
11.40 11.40 3.84 11.7 15.6 7.56
C
rTSH (ng/ml)
44.4 87.9 50.4 50.4 53.7 39.0
1.65 1.26 0.99 1.17 0.66 1.56 1.50
B
B
T 4 (ng/ml)
1.75 0.75 0.21
o 3
1.63
± 1.27 ± 0.45
Case 1 was a 53 year old woman, whose serum was examined he fore and 6 months after radioiodine treatment for clinical hyperthyroidism, which was proven by negative TRH tests and accelerated intrathyroidal radioiodine exchange. After alcohol extraction total T 4 was 225 ngJml and T 3 was 6.7 ngJml before treatment. Without alcohol extraction, T 3 was below the limit of detection (0.2 ng/ml), when correction was made for the assay UB instead for the high VB (45%) of this individual serum.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
No
R. Hehrmann et aL
6 months after treatment the patient was clinically improved although still hyperthyroid. Total T 4 after extraction was now 55 ng/mI and T 3 was 3.3 ng/mI. The T 3 antibody dilution curves of this serum before and 6 months after radioiodine treatment are shown in Figure 4 as compared to an antibody negative con· trol serum. Binding of labelIed T 4 (4%) was not in· creased before and after treatment.
1:
.-
25
50
100
250
500
dilution of "serum
Fil. 4. T 3 antibody determination of case I, demonstrated as % binding of 125J-T3 by different dilutions of patients's serum. o before treatment with radioiodine, clinically hyperthyroid, total T 3 = 6.6 na/ml ~ 6 months after treatment with radioiodine, clinica1ly sUghtly hypothyroid, total T 3 = 3.3 na/mt + Antibody negative controt, normal serum, total T 3 =
1.1 ng/mL
Case 2 is a 40 year old woman with no clinical signs of thyroid dysfunction. T 3 after extraction was 0.7 ng/mI, T 4 was 67 ng/mI, both values being in the lower normal range. Unspecific binding for T 3 was 30.5% at 1: 10 and for T 4 4% at 1: SO dilution. The TSH concentrations of this patient was borderline, but showed exaggerated response to TRH (TSH o = 6.6, TSH 30 = 32 #lU/mI). Case 3 is a 28 year old patient 5 years after treatment with thiamazoie and thyroxine for hyperthy· roidism. She was now clinically euthyroid without treatment. Total T 3 was 5.03 ng/ml, total T 4 was 87 ng/mI after extraetion. Inereased binding of labelIed T 4 could not .be demonstrated, however, 32.7% of labelIed T 3 were bound at 1: 10 dilution She had anormal TSH response to TRH (TSHo = 5.2, TSH 30 = 12.9 #lU/mI). Case 4 is a 30 year old woman with mild elinieal signs of hyperthyroidism. Total T 3 was 23.5 ng/mI and total T 4 was 88 ng/mI after extraetion. Binding of labeUed T 3 was 73.5% at 1: 10 dilution and of T. was 4% at 1: 50 (this patient did not attend our clinic, a TRH test was not performed).
It should be noted, that all of these patients did have variable eoneentrations of anti T 3 autoantibodies, but no T 4 antibodies. Also they had no an· tithyroglobulin antibodies (TRC-test positive between 1:1 and 1:5). Antimicrosomal antibodies have not been determined.
Discussion Previous studies of Nieschillg et al. (1973), Herrmann, Rusche and Krüskemper (1975) and many others, who investigated animals at relatively short intervals after active immunization with hormones suggested neutralization of endogenous hormones by the antibodies produeed, subsequent stimulation by an increase of pituitary hormones leading to markedly elevated levels of the peripheral hormone. Despite the high total hormone concentiations there were °no clinieal signs of hyperfunetion and despite the high antibody titers, there were no signs of hypofunction except for the mild elevation of LH after testosterone immunization. However, Thomeycrajt et al. 1975 demonstrated in a long term study, that LH changes were only transient. This study coneentrated on a late time interval after imrnunization with thyroid hormones and on the quantitation of biological hormone action. For this purpose the determination of oxygen consumption of immunized rats as a parameter of the basal meta· bolie rate and as an index of the thyroid status was chosen using the metabolic eages described. It can clearly be stated, that 6 months after active immunization with T 3 and/or T 4 all animals had cir· culating autoantibodies of measurable titer and in· creased total hormone eoneentrations, whieh were partially excessively high. It may seem to be surprising that the T 3 immunized animals had also slightly inereased T 4 eoneentrations and increased binding of labelIed T 4 indicating small amounts of T 4 antibodies. The T 4 immunized animals had also elevated T 3 levels and T 3 antibodies of low eoncentrations. One explanation could be, that the T 3 conjugates were not free from T 4 contamination and the T 4 conjugates from T 3. However, the rabbits, immunized with the same material had highly speeifie antibodies now used in our RIAs (Hesch and Hüfner 1972, Hehrmllnn and Schneider 1974). It seems more likely, that the rats did not produee highly speeific antibodies. The results ean best be explained by a crossreaction of the 10w affmity T 3 antibodies with T4 and viee versa of about 1-3%. Despite the pre· senee of thyroid hormone autoantibodies and high total hormone eoncentrations, all immunized animals had the same weight development and the same oxygen eonsumption as the eontrols with normal hor· mone eoneentrations and no antibodies. Also they had unchanged rTSH levels as an indicator for thyroid function.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
330
In connection with the various studies of other groups already quoted one can assume the following time course of hormone and antibody production: In a first phase after active immunization antibody concentrations rise and may biologicalty neutralize endogenous hormone with transient decrease of free hormone concentrations. Assuming an intact feedback mechanism this leads to a stimulation of pituitary tropic hormone secretion and thereby to a stimulation of the peripheral gland, exhtbiting histometrical signs of hyperfunction (Nieschlag et al. 1973). Total hormone concentrations rise until alt of the antibody produced is saturated by hormone. This is particularly supported by the fmding, that total hormone concetrations are closely correlated to the antibody titers. At that time TSH or LH concentrations are no longer elevated. Booster injections again lead to antibody production, LH stimulation, further increase of testosterone levels etc. (Thomeycraft et al. 1975). At the time of our investigation the biologicalty ineffeetive secretion of hormone excess by the thyroid after immunization had already stopped, since antibody titers had not been raised by booster injections. A new equilibrium at a higher level of total hormone eoncentration was reached, the animals seemed euthyroid from weight development and general appearance, from oxygen consumption and rTSH coneentrations in eorrelation to histological fmdings. It must be assumed, that free hormone coneentrations, which have not been directly measured in this study, are normal at that time, since neither the central receptor shows any measurable signs of disturbance (normal rTSH) nor does the peripheral receptor (normal oxygen consumption). From these experimental results it can be coneluded that the degree of hormone elevation in antibody positive sera of patients does not give any information on the clinical status of thyroid function in these individuals. It does, therefore, not surprise, that thyroid hormone autoantibodies in connection with high total hormone eoneentrations (if measured after alcohol extraction) can be found in euthyroid, hypothyroid and hyperthyroid patients (Staeheli et al. 1975).
331
Radioimmunological determinations of T 3 and T4 in antibody containing sera are only reliable after alcohol extraction, since without extraction, the individual VB of that serum will be mueh higher than the assay VB and the results obtained will be falsely low or even not measurable. However, the diagnosis cannot be based on the - correet - total hormone eoncentrations either, since the greater part is neutralized by the cireulating antibodies. In fact 2 from our 4 patients were clinicalty euthyroid and case 4 with T 3 excess (23.5 ng/ml) was only slightly hyperthyroid. It is interesting, that case 2 with normal total T3 and T4 and low titer anti-T 3 -antibodies did have a pathological TRH test, indicating, that this patient may become clinically hypothyroid. It seems important to us that we could not detect T4 autoantibodies in human beings. Theoretically one could expect thyroid hormone aotoantibodies being the result of release of thyroglobulin from a diseased gland. This should, if at all, lead to antithyroglobulin and anti T4 antibodies, both of which were not deteetable. T 3 autoantibodies may, therefore, be of other origin than from the hapten thyroglobulin-T3. Conjugation of T 3 to peripheral immunoglobulins under pathological circumstances must be seriously considered as one possibility. The diagnosis of patients with T 3 and/or T4 antibodies must be made on the basis of the clinical fmdings and of the TRH test. From a clinical point of view the existence of thyroid hormone aotoantibodies does not seem to be rare. Since they can only be deteeted, if the individual unspeeific binding of each serum is determined, this should be routinely done for alt sera, that enter a T 3 /T 4 RIA laboratory. Several years ago we have already commented on the importance to determine V.B., because deviation from the blank binding was rather frequent (Hesch, Hüfner, von zur Mühlen and Köbberling 1974). However, these additional determinations would mean an extremely increasing expenditure, so that we claim for these additional investigations at least in all cases, where there are discrepaneies between clinical appearance, T 3 /T 4 concentrations and the results of the TRH test.
Referenees Denckla, W.D.: Minimal oxygen eonsumption in the female rat, same new definitions and measurements. J.AppLPhysiology 29: 2(;3-273 (1970) Denckla, W.D., E. Marcum: Minimal O 2 consumption as an index of thyroid status: Standardization of method. Endocrinology 93: 61-73 (1973) Fraser, R., B.E.C Nordin: The basal metabolie rate during sleep. Laneet I: 532-533 (1955) Harvey, D.G.: The measurement of oxygen eonsumption in small animals. J.Pharmacy Pharmaeology 10: 483-492 (1958) Hehrmann, R., C. Schneider: Der Radioimmunoassay fUr Trijodthyronin und Thyroxin im Serum und seine An-
wendung bei Hyperthyreosen. Radiologe 14: 156-160 (1974) Herrmann, J., H.J. Rusche, M. Berger, H.L. KTÜskemper: Thyroid funetion and triiodothyronine and thyroxine kineties in rabbits immunized with thyroid hormone. Aeta Endocrinol. (Kbh.) 78: 276 (1975) Hesch, R.·D., M. Hüfner: Highly speeific antibodies to triiOdothyronine. Acta BioLMed.Germ. 28: 861-867 (1972) Hesch, R.-D., M. Hüfner, A. von zur Mühlen, J. Köbberling: Radioimmunochemical measurement of thyroid hormones and thyroxine-binding globulin. IAEA-SM- 177/30 (1974) 161
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Anti Thyroid Hormone Autoantibodies under Experimental and Clinica1 Conditions
332
Sigbritt Werner, H. Löw
Holtkamp, D.E., S. Ochs, C. C. Pfeiffer, A.E. Heming: Determination of the oxygen consumption of groups of rats. Endocrinology 56: 93-104 (1955) MitaumJl, T. J. Colucci, L. ShenkmJln, C.S. Hollonder: Rapid simultaneous radioimmunoassay for trüodothyronine and thyroxine in unextraeted serum. Bioehern. Biophys.Res.Comm. 46: 2107-2114 (1972) Nieschlog, E., J. HerrmJlnn, X.H. Usadei, U. Schwedes, K. Schöffling, H.L. Krüskemper: Thyroid hypertrophy and hyperfunetion caused by aetive immunization with thyroid hormones. J.Endocr. 57: 555-556 (1973)
Horm. Metab. Res. 9 (1977) 332-336
© Georg Thieme Verlag Stuttgart
Urinary Excretion of Cyclic AMP and Cyclic GMP in Primary Hyperparathyroidism with Reference to Clinical Signs and Symptoms Sigbritt vyerner and H. Löw Department of Endocrinology and Metabolism, Karolinska Hospital, Stockholm, Sweden Summary 3S patients with surgica1ly proven primary hyperparathyroidims (PHPT) were evaluated with regard to urinary cyclie AMP and cyelie GMP excretion. They showed no signs of bone disease or impaired renal function. Preoperatively, cyelie AMP was increased in 28 patients (80%). Mean excretory value for the whole group was 8.5 ± 0.8 micromoles/g creat/24 hours, compared with controls, 4.2 ± 0.13 micromoles/g creat/24 hours. Cyclic AMP excretion was normalized after surgery.
Urinary cyclic GMP was increased in 14 patients (40%) compared with excretion rates of controls, 0.46 ± 0.03 micromoles/g ereat/24 hours. Cyclie GMP increments persisted postoperatively, although at a lower level. There was an apparent lack of effect of one nucleotide on the other. Clinical, pathological' and biochemical data were analysed and correlated for signifieant assoeiations. The patients demonstrating augmented cyclic GMP excretion rates were not distinguishable from those with normal cyclic GMP production rates with regard to S-calcium, U-calcium, Ucyclie AMP, S-phosphate, cholelithiasis, nephrolithiasis, hypertension or age. One of twelve patients meeting the criteria of normacalcemie PHPT had increased excretion of eyclie AMP and was submitted to parathyroid surgery. Microscopy showed four normal glands. Dur data indicate that the increased excretion of cyelic GMP in urine was not a metabolie efract of the parathyroid hormone, since increments were noted also after reseetion of the parathyroids. Furthermore, it is not likely that the cyelic GMP increments reflected an increased secretion of calcitonin, since 'these increments were noted during hypercalcemia as weil as normocalcemia. The source and cause of produetion of the cyelic GMP remain to be determined. Received: 30 June 1976
Accepted: 1 Dec, 1976
Key-Words: PrimJlry Hyperparathyroidism - Cyclic AMPCyclic GMP
Introduction Patients with primary hyperparathyroidism (PHPT) and nonnal renal function show similar disturbances with regard to calcium, phosphate parameters and parathyroid pathology irregardless of the presence or absence of a long history of repeated renal colics (Lloyd 1968, Wem er, Hjem and Sjöberg 1974). This fmding is of clinical importance, since "asym. ptomatic" PHPT will oe diagnosed in a steadily increasing number due to the use of serum multichannel auto-analyzers. It emphasizes also the fact that disclosing prevalent hypercalcemia will not help in the early diagnosis of PHPT. Patients with PHPT have usually increased excretion of cyclic 3'S'-adenosine monophosphate~yclic AMP) illustrating parathyroid honnone induced augmented adenylate cyclase activity in the kidney (Chose and Aurbach 1967, Kaminsky, Broadus, Hardman, Jones, Ball, Sutherland and Liddie 970, Melson, Chose and Aurbach 1970, Gill and Casper 1971). The analysis of urinary cyclic AMP can be a helpful diagnostic tool to exclude other hypercalcemic states (Kaminsky et al. 1970, Neelon, Drezner, Buch and Lebovitz 1973, Murad and Pak 1972). However, data are sparse showing the value of the cyclic AMP analysis in conflITIling the diagnosis of PHPT in patients with nonnocalcemia and active stone disease (Murad and Pak 1972, Schmidt-Gayk and Röher 1972).
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Nieschlag, E., K.H. Usadei, H.H. Kley, U. Schwedes, K. Schöf/ling, H.L. Krüskemper: A new approach for investigating hypothalamo-pituitary-gonadal and adrenal feedback control mechanism: Active immunization with steroids. Acta Endocrinol. 76: 556-569 (1974) Staeheli, v., M.B. Valloton, A. Burger: Deteetion of human anti-thyroxine and anti-triiodothyronine antibodies in different thyroid conditions. J .Clin.Endocrinol.Metab. 41: 669-675 (1975) Thorneycraft, IH., N.K. Thorneycraft, R.J. Scaramuzzi, ChA. Bloke: Radioimmunoassay of serum LH and testosterone in male rabbits aetively immunized against testosterone. Endocrinology 97: 301-306 (1975) Request for reprints should be adressed to: Dr. R. Hehrmann, Seetion of Clinical Endocrinology, Department of Internal Medicine, Medical School. Hannover, 0.3 Hannover (Germany)
